【作者】 迟文娟；

【导师】 曹远银；

【作者基本信息】 沈阳农业大学 ， 有害生物与环境安全， 2009， 博士

【摘要】 小麦白粉病是我国及东北春麦区的重要小麦病害。小麦白粉病的经济有效防控办法就是首先使用抗病品种,再结合建立在准确预测预报基础上的适时药剂防治。本文围绕这个控制小麦白粉病的原理展开了相关研究。首先研究了2007～2008年东北春麦区及山东、河南、湖北冬麦区小麦白粉病菌(Blumeria graminis f.sp.tritici)种群动态并分析了小麦生产品种(系)的抗白粉病性(基因)。利用ISSR分子标记技术分析了上述地区间的小麦白粉病菌遗传多样性,对病菌毒性多态性进行了聚类分析,比较了小麦白粉病菌遗传多样性、毒性多态性和地域之间的关系,试图为以前报道的“东北春麦区白粉病初菌源来自山东等南部冬麦区”的结论提供DNA分子证据。针对小麦白粉病的ITS区域,应用常规PCR,DAN Dot-blot和Real-time PCR三种方法分别研究了小麦白粉病菌快速分子检测技术,试图为小麦白粉病的流行测报提供新的分子生物学手段。此外,还研究了针对另三种重要小麦病害(纹枯病、散黑穗病和赤霉病)的PCR检测技术。通过上述研究,取得了如下主要进展:1.开展了小麦白粉病菌种群动态与品种抗性(基因)分析。种群动态分析结果表明:从223个菌株中鉴定出61个生理小种。其中在东北春麦区,2007年的的优势小种为17号小种,其出现频率为10.2%;2008年则411号小种出现频率最高,为20.9%;11号、415号、611号和631号小种出现频率也呈现逐年升高的趋势。2008年在山东、河南、湖北冬麦区出现频率最高的为1号小种,出现频率为16.4%。毒性基因分析结果表明,毒性基因V2,V4b,V2+6,V4+8,V12,V13,V16,V18,V20,V21,V22和V23的毒性频率在0.0%～29.9%之间,其对应的抗性基因Pm2,Pm4b,Pm2+6,Pm4+8,Pm12,Pm13,Pm16,Pm18,Pm20,Pm21,Pm22和Pm23的抗性较强,可作为有效的抗病基因加以利用。白粉病菌的联合毒性测定结果表明,147个白粉病菌单孢子堆分离物中含有9个毒性基因组合的出现频率最高,达到15.64%,V1,V3c,V5,V6,V7,V8,V17等毒性基因组合频率也较高,说明病菌在很大程度上可以联合致病。小麦品种抗性(基因)测定结果表明,302份小麦品种中85%的品种为感病,对其中抗性较好的10份材料进行了抗性基因推导分析,表明小麦品种‘保丰104’含抗性基因Pm2+6,品种‘Sunwon85’和‘WF08-182’均含抗性基因Pm5+Pm8,‘京冬8号’和‘西峰20’含抗性基因Pm2+Pm8,‘南大2419’和‘川育55871’含有Pm24+Pm8,‘兰天18’含有与Era相同的抗性基因,‘兰天17’和‘绵阳28’含有未知的抗性较强的基因。2.利用筛选的18条ISSR引物对来自东北春麦区的12个白粉菌株和山东冬麦区的14个菌株进行了遗传多样性分析,构建了26个小麦白粉病菌的遗传聚类分析图。结果表明:条带相似系数在0.56～0.82,并在相似系数0.61处可聚为四类,一类包括13个菌株,分别为辽宁的11号、17号、7号、411号小种,山东的11号、411号小种,河南的35号、11号、711号、331号小种,湖北的11号、31号、21号小种。二类包括2个菌株,分别为山东的611号小种和辽宁的311号小种。三类包括10个菌株,分别为辽宁的431号、55号小种、611号、631号、731号和77号小种,山东的431号、731号小种,湖北的631号、731号小种。四类包括1个菌株,为辽宁的23号小种。26个菌株的病菌毒性分子聚类分析结果表明:条带相似系数在0.52～1.00,在相似系数0.70处可聚为三类,一类包括3个菌株,分别为辽宁的17号小种、辽宁的55号小种和辽宁的77号小种。二类包括2个菌株,为辽宁的7号小种和23号小种,其它的21个菌株归为第三类。以上结果表明了东北与山东两地的小麦白粉病菌间有较高的DNA亲缘关系。3.应用常规PCR、DNA斑点杂交和实时荧光PCR三种现代分子生物学方法对小麦白粉病菌进行了检测研究,结果表明:(1)设计和筛选的常规PCR引物XBFF/R均能得到全部8个小麦白粉病不同小种的PCR产物,其片段长度同为352bp,而9种供试参考病原菌(黄瓜白粉病菌、小麦条锈病菌、小麦叶锈病菌、小麦秆锈病菌、小麦赤霉病菌、小麦纹枯病菌、小麦散黑穗病菌、玉米纹枯病菌及玉米丝黑穗病菌)及对照均未扩增出条带。设计和筛选的DAN斑点杂交探针检测8个白粉小种均表现为阳性,而9种参考病原菌均呈现为阴性反应。设计和筛选的实时荧光PCR引物XBFTF/R及TaqMan探针XBFTP,检测8个小麦白粉病菌小种也均采集到强的荧光信号,dRn值为0.35～0.45,Ct值为15.92～18.53,表现为强阳性扩增,9种参考病原菌及对照均无荧光信号增加,表现为阴性。说明三种方法中设计与筛选的引物及探针对小麦白粉病菌均具有高度特异性,可以作为一种小麦白粉病菌的特异性检测与诊断的手段。(2)三种检测方法的灵敏度的测定与比较结果表明:常规PCR引物XBFF/R检测到的最低DNA模板浓度为10pg,DNA斑点杂交方法最低检测限为1pg,实时荧光PCR最低检测限为1fg。(3)用常规PCR和实时荧光PCR两种方法对温室接种的小麦白粉病进行了检测,并在时间上与自然显症的观察进行了比较,结果表明:在温室条件下,小麦白粉病菌在接种后第5d时开始出现小麦白粉病病斑,而利用常规PCR引物XBFF/R可在接种第3d检测到小麦白粉病菌,比自然显症观察能够提前了2d检测到白粉病菌;实时荧光PCR法可在接种后1d即能检测到小麦白粉病菌,比自然显症观察能够提前了4d检测到白粉病菌,这可为白粉菌的预测或预警检测提供依据,更可为白粉病在流行前制定防治决策赢得宝贵时间。4.本文还通过对小麦纹枯病、小麦散黑穗病、小麦赤霉病病原菌的ITS序列的分析,分别设计和筛选出了三种病原菌的PCR引物,并对三种病害进行了特异性检测,结果表明,小麦纹枯病菌引物XWKF/R对所有供试菌株的DNA进行PCR扩增,只有小麦纹枯病菌扩增出大小为467bp的条带,9种参比病原菌全部均未扩增出条带;小麦散黑穗病菌引物XSHF/R仅能扩增出小麦散黑穗病菌的大小为533bp的条带,所有9种参比病原菌全未扩增出条带。同样,小麦赤霉病菌引物XCMF/R只对小麦赤霉病菌能扩增出大小为401bp的条带而参比病菌全未有条带产生。说明筛选的三对引物对三种病害都各具有特异性。引物灵敏性检测结果表明,引物XWKF/R,XCMF/R分别能够检测出浓度为10pg的小麦纹枯病菌和小麦赤霉病菌,而引物XSHF/R对小麦散黑穗病菌的检测限为1pg。以上结果说明用筛选的三种病原菌的特异性引物可以作为三种病害的检测手段加以利用。更多还原

【Abstract】 Wheat powdery mildew caused by Blumeria graminis f.sp.tritici is one of the most important wheat diseases in our country,esp.in Northeastern Spring Wheat Region.The most effective control way of wheat powdery mildew is to use resistant varieties in combination with right time emergency chemical control based on accurate prediction and forecast.In the present paper all the relevant studies conducted are around this disease control principle.Firstly,race population trend and virulence variation of Blumeria graminis f.sp.tritici in the Northeastern Spring Wheat Region,Shandong,Henan and Hubei Winter Wheat zones in the years 2007 and 2008,and the resistance to the disease contained in wheat varieties(lines) were studied.Secondly,the genetic diversity of wheat powdery mildew,molecular virulence clustering were analyzed and the correlation among molecular diversity,virulence diversity and epidemic zone was analyzed using ISSR method.Thirdly,aimed at the internal transcribed spacer(ITS) of ribosome of Blumeria graminis f.sp.tritici,molecular detection techniques using conventional PCR,DNA Dot-blot,Real-time PCR were studied,specific primers and probes were designed and repeatedly tested,the optimized conditions for detection and diagnoses were conducted.At last,PCR detection techniques to another three important diseases(wheat sheath blight,wheat loose smut and wheat scab) were studied.The main results were as it follows:1.The analyses of population dynamics of the pathogen and the resistance(gene) contained in wheat cultivars were carried out.For the population dynamics,the result was shown that 61 races were met with from 223 isolates tested.In northeastern spring wheat region,race 17 was dominant in the year 2007 with the occurrence frequency being 10.2%.In 2008,the race 411 became most common with an occurrence frequency of 20.9%,and the trend of occurrence frequency for races 11,415,611 and 631 appeared increasing.The prevailing race was race 11 with the frequency of 16.4%in Shandong winter wheat belt in the year 2008.The virulence gene analysis was shown that virulence frequencies for V2,V4b,V2+6,V4+8,V12,V13,V16,V18,V20, V21,V22and V23 were low,distributing between 0.0%~29.9%,indicating the corresponding genes Pm2,Pm4b,Pm2+6,Pm4+8,Pm12,Pm13,Pm16,Pm18,Pm20, Pm21,Pm22,Pm23 were effective and applicable in breeding.The virulence association of 147 isolates was studied and shown that the frequency of the single spore isolates that contain 9 virulent gene association was high,with its frequency being 15.64%.Powdery mildew resistance(gens)in 302 wheat cultivars(lines)was appraised and the effective resistant genes carried in 10 resistant cultivars were inferred.It was indicated that the cultivar ’Baofeng 104’ contained resistant gene(s)Pm2+6,’Sunwon85’ and ’WF 08-182’, Pm5+Pm8,’Jingdog 8’ and ’Xifeng 20’,Pm2+Pm8,and ’Nanda 2419’ and ’Chuanyu 55871’, Pm24+Pm8.’Lantianl8’ had the same resistant gene as ’Era’,’Lantianl7 ’and ’Mianyang 28’ carried resistant genes unknown. 2.The genetic diversity and molecular virulence clustering for the powdery mildew were studied.12 isolates from Northeastern Spring Wheat Region and 14 isolates from Shandong and Henan and Hubei Winter Wheat zones were tested with 18 ISSR primers.Clustering analysis was showed that these isolates were sorted into 4 groups at a similarity of 0.61.Group 1 consisted 13 isolates,races 7,11,17,411 from Liaoning,races 11 and 411 from Shandong, races 11,35,331 and 711 from Henan and races 11,21 and 31 from Hubei.Group 2:race 611 from Shandong and race 311 from Liaoning.Group 3 had 10 races including races 431,55, 611,631 and 731 and 77from Liaoning,races 431 and 731 from Shandong,races 631 and 731 from Hubei.Group 4 contained 1 race:Race 23 from Liaoning.The dendrogram was constructed based on virulence clustering.The similarity of 26 isolates was 0.52～1.00.They could be clustered into 3 groups at the point of similarity 0.70. Group 1 contained race 17,55 and 77 from Liaoning.Group 2,race 7 and 23 from Liaoning. Group 3 included the rest 21 isolates.The above results were indicated that the closed DNA relationship existed between the wheat powdery mildews from Shandong winter area and from Northeastern spring wheat region. 3.Three molecular detection and diagnose techniques based on internal transcribed spacer (ITS)of rDNA of Blumeria graminis f.sp.tritici were studied.The result was shown that by primers or probes designing and screening,and the detection and diagnose conditions optimizing,the primer pair XBFF/R of PCR,the probe of DNA Dot-blot,the real-time primer pair XBFTF/R with TaqMan probe were proven specific and validated with other 9 related plant pathoens as references.With conventional PCR primer pair XBFF/R,the same 352bp bands appeared by amplifying all the DNA samples from the eight races,and no bands appeared by amplifying all the DNA samples from the 9 plant pathogen species(Cucumber powdery mildew,wheat yellow rust,wheat leaf rust,wheat stem rust,wheat scab,wheat sheath blight,wheat loose smut,maize sheath blight and maize head smut)and control.With the probe of DNA Dot-blot,all the eight powdery mildew DNA samples were detected to be positive,and all the rest 9 plant pathogen DNA samples and control were shown negative.With the real-time primer pair XBFTF/R and TaqMan probe,the strong positive signals were obtained from all the eight powdery mildew DNA samples,with the values for dRn:0.35~0.45,Ct:15.92~18.53,and otherwise,no signals increased for all the 9 plant pathogen DNA samples and control.The detection sensitivity test for the three methods was conducted.It was indicated that the detection limits to DNAs of Blumeha graminis f.sp.tritici.were 10pg for the conventional PCR primer pair XBFF/R,1pg for probe of DNA Dot-blot and as low as 1fg for the real-time primer pair XBFTF/R with TaqMan probe.The comparision of the least time needed from powdery midew inoculation to disease dectected or seen was made under greenhouse condition among conventional PCR detection, Real time PCR detection and natural observation.It was shown that under greenhouse condition,the DNAs from powdery mildew infected wheat leaves were detected 3 days after inoculation with conventional PCR assay and only one day with real-time PCR,the disease lesions were seen with naked eyes 5days after inoculation.indicating that the disease after inoculation was detected two days earlier with conventional PCR and 4days earlier with real time PCR than natural observation,respectively. 4.Molecular detection techniques based on ITS were also used to detect wheat diseases; wheat sheath blight:Rhizoctonia cerealis,wheat loose smut:Ustilago nuda and wheat scab:Fusarium graminearum.3 primer pairs XWKF/R,XSHF/R and XCMF/R were designed and tested.It was shown that all the primer pairs were each unique and highly sensitive to the corresponding target pathogen.更多还原