The Distribution And Toxin Type Of Fusarium Graminearum Clade In Anhui

Wheat scab is one of the important worldwide diseases of wheat. In this article,the major flora distribution of Fusarium graminearum Schw including F. asiaticumand F. graninearum in Anhui is studied by the molecular method of PCR-RFLP.Thereason of why does it has this distribution regular pattern is searched from twodirections.One of them is the relation between the distribution regular pattern and theprevious crops, the other one is the effection of Geographic distance. At the same time,the types of toxin produced by Fusarium graminearum Schw is also checked usingthe molecular technology. In order to quanlitify the content of toxin produced byFusarium graminearum Schw in kernels, the amount of mycelium in kernel isdetected by RealTime-PCR refering to the amount of toxin.With the porpurses tocontrol the desease and achieve the foodstuff bumper harvest,the research is launchedorderly.1. Molecular identification of Fusarium graminearum Schw including F.asiaticum and F. graninearum by PCR-RFLP inAnHuiAll of the31disease ear samples were gained from AnHui in this study. TheFusarium graminearum Schw strains were seperated and purified from the diseaseears. By using the method of CTAB, the DNA of all samples were extracted.The disease ears cover the area of AnHui.For example,Taihe, Guoyang,Lixing,Maoji,Fengtai,Yingshang,Fuyang,Mengcheng,Guoyang,Huaiyuan,Suzhou,Dangshan,Chaohu,Dingyuan,Liuan,Shouxian,Jinzhai,Huoqiu,Changfeng,Mingguang,Hanshan,Hengyang,Xuanzhou,Tongling,Qianshan,Huaining,Anqing,Nanling,Fanchang,Wuhu,Xingmaqiao.The product of about200bp by PCR is obtained. Dealed with restriction enzymeof Ecoi V and Sty Ⅰ,the results can be distinguished by the length of product afterrestriction enzyme trement.In the result of this study,only4strains fromShouxian,Dangshan,Mengcheng,Anqing appears to be F. graninearum,the other oneswere F. asiaticum.The same to the dete of Hu-yingchun, bounded by the YangtzeRiver, F. graninearum majors in northen China and F. asiaticum majors in southenChina.2. The analysis of the relation between flora distribution and the previouscrops (wheat and cron)Large samples(30samples each point) were taken from Mengcheng(previous crop cron), Huaiyuan(previous crop rice),Middle area of Yingshang(previous cropcron and rice),15kilometres to the middle of Yingshang,30kilometres to the middleof Yingshang.All of the strains were purified to PDA.The DNA was extracted byCTAB.The product of about200bp by PCR was obtained. Dealed with restrictionenzyme of Ecoi V and Sty Ⅰ,the results could be distinguished by the length ofproduct after restriction enzyme trement.In the end,we found that there were several strains of F. graninearum inMengcheng(previous crop cron).It had the rate of10%of samples.All of the strainsfrom Huaiyuan(previous crop rice) appeared to be F. asiaticum. The results displaiedthe previous crop effected the flora distribution to some extent.The same to the resultof Middle area of Yingshang(previous crop cron and rice).Seveal strains of F.graninearum were found in the area with previous crop cron. While,after checkingthe rate of F. graninearum among Middle area of Yingshang(previous crop cron andwheat),15kilometres to the middle of Yingshang,30kilometres to the middle ofYingshang,we came to the conclusion that Geographic distance had no significantaffection to the flora distribution.3. Molecular identification of toxin produced by Fusarium graminearum complexAll of the31disease ear samples were gained from AnHui in this study.TheFusarium graminearum Schw strains were seperated and purified from the diseaseears. By using the method of CTAB, the DNA of all samples were extracted.The disease ears cover the area of AnHui.For example,Taihe, Guoyang,Lixing,Maoji,Fengtai,Yingshang,Fuyang,Mengcheng,Guoyang,Huaiyuan,Suzhou,Dangshan,Chaohu,Dingyuan,Liuan,Shouxian,Jinzhai,Huoqiu,Changfeng,Mingguang,Hanshan,Hengyang,Xuanzhou,Tongling,Qianshan,Huaining,Anqing,Nanling,Fanchang,Wuhu,Xingmaqiao.Primer pairs:ToxP1(5’-GCCGTGGGRTAAAAGTCAAA-3’)ToxP2(5’-TGACAAGTCCGGTCGCACTAGC-3’)According to the PCR product length,we could know that the strains whichgenerate toxin type of NIV has fragment of360bp and DON300bp.Primer pairs:Tri303F(5’-GATGGCCGCAAGTGGA-3’)Tri303R(5’-GCCGGACTGCCCTATTG-3’) We could judge the toxin type of strain to be3-DON,if we got the fragment of586bp.Primer pairs:Tri315F(5’-CTCGCTGAAGTTGGACGTAA-3’)Tri315R(5’-GTCTATGCTCTCAACGGACAAC-3’)We could judge the toxin type of strain to be15-DON,if we got the fragment of864bp.Following the methods,we got the result that the strains fromWuhu,Qianshan,Chaohu,Huoqiu,Taihe and Tongling appear to produce toxin ofNIV.The strains from Mengcheng,Dangshan,Shouxian,Anqing produce toxin of15-DON.The others generate3-DON.An interesting phenomenon was found that theones produce toxin of15-DON were F. graninearum only.4. Making standard curve for checking the DNA of Fusarium graminearum Schw byRealTime-PCRThe DNAof strain was extracted by CTAB.Primer pairs-Tri5:Tri5F5’-AGCGACTACAGGCTTCCCTC-3’Tri5R5’-AAACCATCCAGTTCTCCATCTG-3’With the SYBR Priemix Ex Taq TM II (Tli RNaseH Plus，TaKaRa) system,wegot the standard curve equation: y=-4.6953lg(x)+6.4496R2=0.9982.5. The identification of the strain from NanlingA special strain was found from Nanling.The molecular identification waslaunched by ITS and PCR-RFLP.Each result displaied it belonged to Fusariumasiaticum.The mycelial growth rate, pigment,sporulation,and pathogenicity were checkedand compared between natural strains.Its dete were lower than common ones.