Rapid Identification of Main Pathogen Fusarium pseudograminearum of Wheat Crown Rot Using Site-specific PCR Based on EF-1α Sequence
LIU Guo-Xia1,*, TAN Qing-Qing1, QI Jun-Shan2, WANG Fu-Yu3, CHEN Xue-Yan1, FAN Yang-Yang1, HU Yue1, BU Xun1, ZHANG Quan-Fang1,*
1 Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, China; 2 Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan 250100, China; 3 Jining Academy of Agricultural Sciences, Jining 272031, China
Abstract：Wheat stem rot (WCR) is an important disease affecting wheat yield, Fusarium pseudograminearum is the main pathogen. The rapid and accurate identification of pathogens is significant for making reasonable control measures and resistant varieties breeding. In this study, elongation factor 1α (EF-1α) gene sequences of common Fusarium spp. were compared and specific primers F.pseudo-F3/R1 for F. pseudograminearum were designed, touchdown PCR were used to specifically amplify a 334 bp band from F. pseudograminearum, and no band amplified in F. graminearum, F. tricinctum, F. proliferatum, F. incarnatum and F. oxysporum. Wheat samples collected from Jining and Jiyang in Shandong province in 2019 and 2020 were detected using the site-specific PCR method which was just developed to test its validity. It was found that 28 fungi strains were isolated from infected samples from Jining in 2019, in which F. pseudograminearum was amplified with aimed band of 334 bp using primers F.pseudo-F3/R1, while no band from other fungi including F. graminearum, F. equiseti, Alternaria alternate, Bipolaris sorokiniana and Rhizoctonia cerealis. Samples were also collected from Jining in 2020 and DNA isolated directly from wheat stem with symptom of crown rot, then amplified using primers F.pseudo-F3/R1 and got a 334 bp aimed band of F. pseudograminearum, while other samples with symptom of root rot, leaf rust, powdery mildew or white-headed got no amplification band. The fungi isolated from stem of samples used above from Jining in 2020 were identified by sequencing analysis, and the result showed that F. pseudograminearum and F. graminearum existed in crown rot wheat sample, while F. graminearum and R.cerealis existed in other samples. The wheat stems of artificially and naturally infected by F. pseudograminearum from Jiyang in 2020 were amplified using primers F.pseudo-F3/R1 and got a 334 bp aimed band of F. pseudograminearum. The above results indicated that the specific PCR method could be used to detect F. pseudograminearum in stem directly, which provides a new method to rapidly identify F. pseudograminearum.
刘国霞, 谭晴晴, 齐军山, 王福玉, 陈雪燕, 范阳阳, 胡悦, 步迅, 张全芳. 基于EF-1α序列位点特异性PCR快速鉴定小麦茎基腐病优势病原菌假禾谷镰孢菌[J]. 农业生物技术学报, 2021, 29(5): 985-994.
LIU Guo-Xia, TAN Qing-Qing, QI Jun-Shan, WANG Fu-Yu, CHEN Xue-Yan, FAN Yang-Yang, HU Yue, BU Xun, ZHANG Quan-Fang. Rapid Identification of Main Pathogen Fusarium pseudograminearum of Wheat Crown Rot Using Site-specific PCR Based on EF-1α Sequence. 农业生物技术学报, 2021, 29(5): 985-994.
[1] 代君丽, 于巧丽, 袁虹霞, 等. 2011. 河南省小麦黑胚病菌的分离鉴定及致病性测定[J]. 植物病理学报, 41(3): 225-231. (Dai J L, Yu Q L, Yuan H X, et al.2011. Isolation, identification and pathogenicity of the pathogens of wheat black point in Henan province[J]. Acta Phytopathologica Sinica, 41(3): 225-231.) [2] 李伟, 陈莹, 张晓祥, 等. 2011. 小麦茎基褐腐病病原菌组成及其致病力研究[J]. 麦类作物学报, 31(1): 170-175 (Li W, Chen Y, Zhang X X, et al.2011. Pathogenic fungi of wheat brown foot rot and their pathoginicity in China[J]. Journal of Triticeae Crops, 31(1): 170-175.) [3] 史亚娟, 王英志, 王振跃, 等. 2016. 假禾谷镰刀菌LAMP快速检测方法的建立[J]. 植物病理学报, 46(4): 566-568. (Shi Y J, Wang Y Z, Wang Z Y, et al.2016.Loop mediated isothermal amplification assay for sensitive and rapid detection of Fusarium pseudograminearum[J]. Acta Phytopathologica Sinica, 46(4): 566-568.) [4] 王婧, 翟伟卜, 高环, 等. 2017. 链格孢引起的病害严重危害农作物生产并危及农产品安全[J]. 植物保护, 43(4): 9-15. (Wang J, Zhai W B, Gao H, et al.2017. Serious damage to crop production caused by Alternaria diseases and the safety of agricultural product[J]. Plant Protection, 43(4): 9-15.) [5] 吴斌, 郭霞, 张眉, 等.2018. 鲁西南地区小麦茎基腐病病原菌鉴定及其致病力分析[J]. 麦类作物学报, 38(3): 356-365. (Wu B, Guo X, Zhang M, et al.2018. Identification and pathogenicity of pathogens associated with wheat crown rot in the southwest of Shandong Province[J]. Journal of Triticeae Crops, 38(3): 356-365.) [6] 易铭, 梁嘉俊, 史建, 等. 2017. 采用EF-1α序列分析法对苜蓿根腐病病原菌-锐顶镰刀菌的鉴定[J]. 草业学报, 26(2): 61-68. (Yi M, Liang J J, Shi J, et al.2017. Identification of Fusaruim acuminatum isolated from Medicago sativea root using the EF-1α sequence analysis method[J]. Acta Prataculturae Sinic, 26(2): 61-68.) [7] 张德珍, 李鹏昌, 陈晓霞, 等. 2016. 山东省小麦根腐病病原菌的分离鉴定[J]. 植物保护学报, 43(2): 233-240. (Zhang D Z, Li P C, Chen X X, et al.2016. Isolation and identification of the pathogens causing wheat common root rot in Shandong Province[J]. Journal of Plant Protection, 43(2): 233-240.) [8] Al-Hatmi A M, van den Ende A H, Stielow J B, et al.2016. Evaluation of two novel barcodes for species recognition of opportunistic pathogens in Fusarium[J]. Fungal Biology, 120(2): 231-245. [9] Aoki T, O'Donnell K.1999. Morphological and molecular characterization of Fusarium pseudograminearum sp. nov., formerly recognized as the group 1 population of F. graminearum[J]. Mycologia, 91(4): 597-609. [10] Chakraborty S, Liu C J, Mitter V, et al.2006. Pathogen population structure and epidemiology are keys to wheat crown rot and Fusarium head blight management[J]. Australasian Plant Pathology. 35(6): 643-655. [11] Cook R J.1980. Fusarium foot rot of wheat and its control in the pacific northwest[J]. Plant Disease. 64(12): 1061-1066. [12] Dehghanpour-Farashah S, Taheri P, Falahati-Rastegar M.2020. Identification and pathogenicity of Fusarium spp., the causal agent of wheat crown and root rot in Iran[J]. Journal of Plant Pathology, 102(1): 143-154. [13] Ji L J, Kong L X, Li Q S, et al.2016. First report of Fusarium pseudograminearum causing fusarium head blight of wheat in Hebei province, China[J]. Plant Disease, 100(1): 220. [14] Li H L, Yuan H X, Fu B, et al.2012. First report of Fusarium pseudograminearum causing crown rot of wheat in Henan Province, China[J]. Plant Disease, 96(7): 1065. [15] Kazan K, Gardiner D M.2018. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: Recent progress and future prospects[J]. Molecular Plant Pathology. 19(7): 1547-1562. [16] Kvas M, Marasas W F O, Winglfield B D, et al.2009. Diversity and evolution of Fusarium species in the Gibberella fujikuroi complex[J]. Fungal Diversity, 34: 1-21. [17] O'Donnell K, Kistler H C, Tacke B K, et al.2000. Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab[J]. Proceedings of the National Academy of Sciences of the USA. 97(14): 7905-7910. [18] Schoch C L, Seifert K A, Huhndorf S, et al.2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi[J]. Proceedings of the National Academy of Sciences of the USA, 109(16): 6241-6246. [19] Smiley R W, Patterson L M.1996. Pathogenic fungi associated with Fusarium foot rot of winter wheat in the semiarid Pacific Northwest[J]. Plant Disease. 80(8): 944-949. [20] Stielow J B, Lévesque C A, Seifert K A, et al.2015. One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes[J]. Persoonia. 35: 242-263. [21] White T J, Bruns T D, Lee S, et al.1990. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics[M]. New York: Academic Press, NY. pp: 15-322. [22] Williams K J, Dennis J I, Smyl C, et al.2002. The application of species-specific assays based on the polymerase chain reaction to analyse Fusarium crown rot of durum wheat[J]. Australasian Plant Pathology, 31: 119-127. [23] Xu F, Yang G, Wang J, et al.2018. Spatial distribution of root and crown rot fungi associated with winter wheat in the North China plain and its relationship with climate variables[J]. Frontiers Microbiology. 25(9): 1054. [24] Zhang P, Deng Y, Yang X, et al.2017. Pathogen identification of wheat crown rot and control effects of different fungicides[J]. Plant Diseases and Pests, 8(3): 12-15. [25] Zhou H, He X, Wang S, et al.2019. Diversity of the Fusarium pathogens associated with crown rot in the Huanghuai wheat growing region of China[J]. Environmental Microbiology, 21(8): 2740-2754.
