Abstract：Crown rot caused by Fusarium pseudograminearum is one of the main devastating diseases in wheat (Triticum aestivum) production. Studying the interaction mechanism between F. pseudograminearum and wheat varieties is the most economical and effective measures to control of wheat crown rot. The objectives of this study are to optimize the Agrobacterium tumefaciens-mediated transformation (ATMT) technology system of F. pseudograminearum, and obtain the transformant strains of F. pseudograminearum (WZ-8A) successfully transformed with with the green fluorescent protein (GFP). Firstly, the transformation conditions were optimized, and then the GFP gene was transformed into the conidia of F. pseudograminearum strain WZ-8A using the A. tumefaciens strain carrying plasmid pCAM-GFP-Hyg. After transformation, transformants randomly collected were screened and identified through the analysis of the hygromycin B (hyg) resistance, as well as using the fluorescence microscopy techniques. A pathogenicity detection was subsequently conducted. The results showed that a satisfactory transformation with a efficiency of 42 transformants per 1×106 spores could be achieved in a sytem of 1×106 spores per milliliter of F. pseudograminearum spore suspension which were co-cultured with Agrobacterium cells under the culture in the presence of co-culture medium containing CaCl2 at 2.6×10-2 g/L at 28 ℃ for 2 d. The transformants was stable when grown on hygromycin B-free PDA medium for 5 generations. The transformants were found to be hyg-positive by PCR amplification and Southern blot analysis, and to be GFP-positive through the detection using fluorescence microscopy, compared with the wild-type strain of WZ-8A. The subsequent test showed that the transformants really did not lose the pathogenicity. It thus be concluded that the T-DNA bearing GFP gene is successfully inserted into the genome of F. pseudograminearum with the optimized system mediated by A. tumefaciens. The optimized genetic transformation sysytem mediated by A. tumefaciens in F. pseudograminearum could be used for the studies on the pathogenic mechanisms as well as the resistance mechanisms in wheat cultivars.
[1] Bekker C D, Veluw G J V, Vinck A, et al. 2011. Heterogeneity of aspergillus niger microcolonies in liquid shaken cultures[J]. Applied & Environmental Microbiology, 77(4): 1263-1267.[2] Burgess L W, Backhouse D, Summerell B A, et al. 2001. Crown rot of wheat[C]//. Paul E. (eds.) Fusarium, Nelson Memorial Symposium. pp. 271-294.[3] Cardoza V, Stewart C N. 2003. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola ( Brassica napus L.) from hypocotyl segment explants[J]. Plant Cell Reports, 21(6): 599-604.[4] Chakraborty S, Liu C J, Mitter V, et al. 2006. Pathogen population structure and epidemiology are a key to wheat crown rot and Fusarium head blight management[J]. Plant Pathology Australasian Plant Pathology, 35(6): 643-655.[5] Cormack B. 1998. Green fluorescent protein as a reporter of transcription and protein localization in fungi[J]. Current Opinion in Microbiology, 1(4): 406-410.[6] de Groot M J, Bundock P, Hooykaas P J, et al. 1998. Agrobacterium-mediated transformation of filamentous fungi[J]. Nature Biotechnology, 16(9): 839-842.[7] Jansen C, Wettstein D V, Sch?fer W, et al. 2005. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum[J]. Proceedings of the National Academy of Sciences of the United States of America, 102(46): 16892-16897.[8] Li H L, Yuan H X, Fu B, et al. 2012. First report of Fusarium pseudograminearum causing crown rot of wheat in Henan, China[J]. Plant Disease, 96(7): 1065-1065.[9] Mullins E D , Chen X , Romaine P, et al. 2001. Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer[J]. Phytopathology 91(91), 173-180.[10] Murray M G, Thompson W F. 1980. Rapid isolation of high molecular weight plant DNA[J]. Nucleic Acids Research, 8(19): 4321-4325.[11] Poole G J, Smiley R W, Paulitz T C, et al. 2012. Identification of quantitative trait loci (QTL) for resistance to Fusarium crown rot (Fusarium pseudograminearum) in multiple assay environments in the Pacific Northwestern US[J]. Theoretical and Applied Genetics, 125(1): 91-107.[12] Ramos B, López G, Molina A. 2015. Development of a Fusarium oxysporum f. sp. melonis, functional gfp fluorescence tool to assist melon resistance breeding programmes[J]. Plant Pathology, 64(6): 1349-1357.[13] Rho H S, Kang S, Lee Y H. 2001. Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea[J]. Molecules & Cells, 12(3): 407-411.[14] S?rensen L Q, Lys?e E, Larsen J E, et al. 2014. Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system[J]. Bmc Molecular Biology, 15(1): 241-244.[15] Sakaguchi A, Miyaji T, Tsuji G, et al. 2008. Kelch repeat protein clakel2p and calcium signaling control appressorium development in Colletotrichum lagenarium[J]. Eukaryotic Cell, 7(1): 102-111.[16] Shao C, Yin Y, Qi Z, et al. 2015. Agrobacterium tumefaciens-mediated transformation of the entomopathogenic fungus Nomuraea rileyi[J]. Fungal Genetics & Biology, 83: 19-25.[17] 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.[18] Song J Y, Lu S F, Chen Z Z, et al. 2006. Genetic Transformation of Populus trichocarpa Genotype Nisqually-1: A Functional Genomic Tool for Woody Plants[J]. Plant and Cell Physiology, 47(11): 1582-1589.[19] Spellig T, Bottin A, Kahmann R. 1996. Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungus Ustilago maydis[J]. Molecular Genetics and Genomics, 252(5): 503-509.[20] Stephanie, Widyastuti U, Wiyono S. 2015. Introduction of the serine green fluorescent protein (sgfp) gene into Pyricularia grisea race dc4 isolated from Digitaria ciliaris using Agrobacterium tumefaciens-mediated genetic transformation[J]. Biotropia, 22(1): 73-79.[21] Suelmann R, Sievers N, Fischer R. 1997. Nuclear traffic in fungal hyphae: in vivo study of nuclear migration and positioning in Aspergillus nidulans[J]. Molecular Microbiology, 25(4): 757-769.[22] Tsuji G, Fujii S, Fujihara N, et al. 2003. Agrobacterium tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium[J]. Journal of General Plant Pathology, 69(4): 230-239.[23] van Wyk P S, Los O, Pauer G D C, et al. 1987. Geographic distribution and pathogenicity of Fusarium species associated with crown rot of wheat in the Orange Free State[J], South Africa. Phytophylactica, 19: 271-274.[24] Zhang P, Xu B, Wang Y, et al. 2008. Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the fungus Penicillium marneffei[J]. Mycological Research, 112(8): 943-949.[25] 方丽, 任海英, 茹水江, 等. 2009. 根癌农杆菌介导的甜瓜遗传转化[J]. 浙江农业学报, 21(3): 211-214. (Fang L, Ren H Y, Ru S J, et al. 2009. Agrobacterum tumefaciens-mediated genetic transformation in melon[J]. Acta Agricalturae Zhejiangensis, 21(3): 211-214.)[26] 刘立全, 杨剑超, 张衡, 等. 2009. 根癌农杆菌介导的植物遗传转化研究[J]. 现代农业科技, (11): 143-143. (Liu L Q, Yang J C, Zhang H, et al. 2009. Knowledge of Plants Genetic Transformation by Agrobacterium[J]. Modern Agricultural Science and Technology, 11: 143-143.)[27] 毛超, 戴青冬, 汪军, 等. 2013. 香蕉枯萎病菌4号生理小种农杆菌介导遗传转化体系的建立及T-DNA插入突变体的筛选[J]. 南方农业学报 44(12), 1985-1991. (Mao C, Dai Q D, Wang J, et al. 2013. Establishment of ATMT Fusarium oxysporum f.sp. cubense race 4 effective transformation system and screening of the T-DNA insertional mutants[J]. Journal of Southern Agriculture, 44(12): 1985-1991.)[28] 杨云, 贺小伦, 胡艳峰, 等. 2015. 黄淮麦区主推小麦品种对假禾谷镰刀菌所致茎基腐病的抗性[J]. 麦类作物学报 35(3), 339-345. (Yang Y., He X. L., Hu Y.F., et al. 2015. Resistance of Wheat Cultivars in Huang Huai Region of China to Crown Rot Caused by Fusarium pseudograminearum[J]. Journal of Triticeae Crops, 35(3): 339-345.)[29] 周海峰, 杨云, 牛亚娟, 等. 2014. 小麦茎基腐病的发生动态与防治技术[J]. 河南农业科学，(5):114-117. (Zhou H F, Yang Y, Niu Y J, et al. 2014. Occurrence and control methods of crown rot of wheat[J]. Journal of Henan Agricultural Sciences, (5): 114-117.)
