Establishment of a Rapid LAMP Detection Assay for Fusarium oxysporum f. sp. niveum
YANG Ke1,2, XIAO Ji-Ling2, ZHANG Yi2, WEI Lin3, LIANG Zhi-Huai1,2,*
1 Longping Branch, Graduate School of Hunan University, Changsha 410125;
2 Hunan Agricultural Biotechnology Research Institute, Changsha 410125;
3 Hunan Institute of Plant Protection, Changsha 410125
Abstract：Fusarium wilt of watermelon caused by Fusarium oxysporum f. sp. niveum (FON) is the main disease of watermelon across all major watermelon-growing regions in China. Therefore, the establishment of a rapid detection method of FON is quite important for the diagnosis, prevention and control of Fusarium wilt of watermelon. In this study, using the specific DNA sequences of FON as the target, a set of sensitive and specific loop-mediated isothermal amplification (LAMP) primers were designed, and combined with the rapid DNA extraction method, a rapid LAMP detection assay for FON was established. The results of primer specificity experiments showed that only FON-0, FON-1 and FON-2 showed yellow-green color for LAMP detection, and the agarose gel electrophoresis results of LAMP products showed trapezoidal bands, indicating that the primers had high specificity. The sensitivity detection results showed that the lower limit of detection of the LAMP assay for FON-1 genomic DNA was 10 pg/μL, and for FON-1 content in plants was 5×102 spores/g. The results of potting experiments showed that the assay could make a correct judgment on whether watermelon seedlings were infested by FON, using root tissues as samples. In this assay, from the beginning of DNA extraction to the detection results, only about 90 min was needed, and the pathogenic mycelia, the tissues of the plants could be used as detection materials. The results of LAMP reaction were discernible to the naked eye, with simple operation, strong specificity, high sensitivity, stability and wide applicability. It provides technical support for rapid molecular diagnosis of FON in the field.
[1] 曹月霞, 凌键, 谢丙炎, 等. 2015. 西瓜枯萎病的快速检测与鉴定[J]. 生物技术通报, 31(7): 58-63.
(Cao Y X, Ling J, Xie B Y, et al.2015. Rapid detection and identification of Fusarium oxysporum f. sp. niveum[J]. Biotechnology Bulletin, 31(7): 58-63. )
[2] 蓝江林, 苏明星, 葛慈斌, 等. 2007. 尖孢镰刀菌的分布与西瓜枯萎病病程的相关性[J]. 中国农学通报, 23(12): 302-305.
(Lan J L, Su M X, Ge C B, et al.2007. Relativity between the distribution of Fusarium oxysporum and the course of disease of wilt disease of watermelon[J]. Chinese Agricultural Science Bulletin, 23(12): 302-305. )
[3] 罗中钦, 程琳, 张茜. 2015. 丝状真菌PCR模板DNA的快速制备方法[J]. 生物技术通报, 31(9): 79-83.
(Luo Z Q, Cheng L, Zhang X.2015. A rapid method of preparing DNA template of filamentous fungi for PCR amplification[J]. Biotechnology Bulletin, 31(9): 79-83. )
[4] 宋丰顺, 倪大虎, 陆徐忠, 等. 2007. 水稻单粒种子DNA的快速制备及PCR分析[J]. 安徽农业科学, 35(21): 6371-6372.
(Song F S, Ni D H, Lu X Z, et al.2007. A quick DNA extraction method from single rice seeds[J]. Journal of Anhui Agricultural Science, 35(21): 6371-6372. )
[5] 汪秀峰, 杨剑波, 向太和, 等. 2002. 一种叶片直接用作PCR扩增的新方法及其应用[J]. 中国水稻科学, 16(1): 61-70.
(Wang X F, Yang J B, Xiang T H, et al.2002. A new method for PCR reaction from alkali-treated rice leaf tissues[J]. Chinese Journal of Rice Science, 16(1): 61-70.)
[6] 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 Y, 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. )
[7] 张兴平, 王鸣. 1991. 我国西瓜枯萎病生理小种分化研究初报[J]. 中国瓜菜, 1: 39-43.
(Zhang X P, Wang M.1991. Preliminary report on different Fusarium Wilt race in China[J]. China Cucurbits and Vegetables, 1: 39-43.)
[8] Duan Y B, Ge C Y, Zhang X K, et al.2014. Development and evaluation of a novel and rapid detection assay for Botrytis cinerea based on loop-mediated isothermal amplification[J]. PLOS ONE, 9(10): e111094.
[9] Fenn K, Blaxter M.2006. Wolbachia genomes: Revealing the biology of parasitism and mutualism[J]. Trends in Parasitology, 22(2): 60-65.
[10] Jiang C H, Yao X F, Mi D D, et al.2019. Comparative transcriptome analysis reveals the biocontrol mechanism of Bacillus velezensis F21 against Fusarium wilt on watermelon[J]. Frontiers in Microbiology, 10: 652.
[11] Karthik K, Rathore R, Thomas P, et al.2014. New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination[J]. MethodsX, 1: e137-e143.
[12] Keinath A P, DuBose A B, Katawczik M M, et al.2020. Identifying races of Fusarium oxysporum f.sp. niveum in south carolina recovered from watermelon seedlings, plants, and field soil[J]. Plant Disease, 104(9): 2481-2488.
[13] Kubicek C P, Steindorff A S, Chenthamara K, et al.2019. Evolution and comparative genomics of the most common Trichoderma species[J]. BMC Genomics, 20: 485.
[14] Li J M, Fokkens L, Dam P V, et al.2020. Related mobile pathogenicity chromosomes in Fusarium oxysporum determine host range on cucurbits[J]. Molecular Plant Pathology, 21(6): 761-776.
[15] Li J, Mahajan A, Tsai M D.2006. Ankyrin repeat: A unique motif mediating protein-protein interactions[J]. Biochemistry, 45(51): 15168-15178.
[16] Li L, Zhang S Y, Zhang C Q.2019. Establishment of a rapid detection method for rice blast fungus based on one-step loop-mediated isothermal amplificationm (LAMP)[J]. Plant Disease, 103(8): 1967-1973.
[17] Liu L, Wang C L, Peng W Y, et al.2015. Direct DNA extraction method of an obligate parasitic fungus from infected plant issue[J]. Genetics and Molecular Research, 14(4): 18546-18551.
[18] Mosavi L K, Cammett T J, Desrosiers D C, et al.2004. The ankyrin repeat as molecular architecture for protein recognition[J]. Protein Science, 13(6): 1435-1448.
[19] Notomi T, Okayama H, Masubuchi H, et al.2000. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 28(12): E63.
[20] Ordonez N, Seidl M F, Waalwijk C, et al.2015. Worse comes to worst: Bananas and panama disease-when plant and pathogen clones meet[J]. PLoS Pathogens, 11(11): e1005197.
[21] Shan L Y, Haseeb H A, Zhang J, et al.2019. A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of toxigenic Fusarium temperatum in maize stalks and kernels[J]. International Journal of Food Microbiology, 291: 72-78.
[22] Siemonsmeier A, Hadersdorfer J, Neumüller M, et al.2019. A LAMP protocol for the detection of 'Candidatus Phytoplasma Pyri', the causal agent of pear decline[J]. Plant Disease, 103(6): 1397-1404.
[23] Sun Y, Wang Y, Han L R, et al.2017. Antifungal activity and action mode of cuminic acid from the seeds of Cuminum cyminum L. against Fusarium oxysporum f.sp. niveum(FON) causing fusarium wilt on watermelon[J]. Molecules, 22(12): 2053.
[24] Wang Y, Wang C W, Wang Y, et al.2020. Development of loop-mediated isothermal amplification (LAMP) assay for rapid detection of Fusarium proliferatum causing ear and kernel rot on maize[J]. Crop Protection, 132:105142.
[25] Willem G. E. J, Schoonen, Walter M. A. Westerink, et al.2005. Cytotoxic effects of 100 reference compounds on Hep G2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I mechanistic assays on ROS, glutathione depletion and calcein uptake[J]. Toxicology in Vitro, 19(4): 505-516.
[26] Zhang M, Xu J H, Liu G, et al.2015. Characterization of the watermelon seedling infection process by Fusarium oxysporum f. sp. niveum[J]. Plant Pathology, 64(5): 1076-1084.
