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混合种植控制小麦白粉病及小麦白粉菌ESTs-SSR引物开发

Use of Cultivar Mixtures for Powdery Mildew Management in Wheat and Exploitation of Wheat Powdery Mildew EST-SSRs

【作者】 许韬

【导师】 段霞瑜;

【作者基本信息】 中国农业科学院 , 植物病理学, 2012, 硕士

【摘要】 小麦白粉病是由禾本科布氏白粉菌小麦专化型(Blumeria graminis f.sp.tritici)引起的危害小麦作物的世界性病害。目前主要采取抗病育种和化学防治进行防控。利用品种混合可以更安全、有效、持久地控制白粉病,选用了八个抗病性不同,遗传背景差异较大的小麦品种矮抗58、新麦19、郑麦9694、众麦2号、周麦18、郑农16、郑麦004和轮选987,于2010~2011年在河北廊坊进行田间多样性布局,同时选用了五个抗病性不同的小麦品种矮抗58、新麦19、众麦2号、周麦18、郑农16,在河南新乡试验基地进行类似实验。研究结果如下:对2010~2011年度的河北廊坊的AUDPC进行分析表明:三、四、五品种混种对白粉病的平均病害防治效应分别为为9.91%、31.15%、11.53%;所有多品种混种处理的防效以四品种混种最好。所有防效中以抗感搭配的防效最好,抗性品种所占比例越高,效果越好。对2010-2011年度的河南新乡实验区的病情指数值进行分析表明:旗下叶法所得两、三、四/五品种混种对白粉病的平均病害防效分别为16.07%、3.22%、27.75%。九级法所得两、三、四/五品种混种对白粉病的平均病害防效分别为4.08%、-3.13%、12.18%。对2010~2011年度的河北廊坊的产量和蛋白质进行分析表明:三、四、五、六品种混种的30穗重相对增加率分别为5.07%,4.39%,4.18%,3.24%;蛋白质相对增产率分别为2.57%,3.51%,0.16%和3.44%。对2010~2011年度的河南新乡的产量和蛋白质进行分析表明:二、三、四品种的产量相对增加率分别为1.84%、2.96%和2.70%;蛋白质相对增加率分别为0.69%、0.20%和-1.12%。除新乡实验蛋白质含量增量外,都是以三品种和四品种的效果较好。进一步研究还发现,一些增量比较显著的处理中有几个共同的品种。而增量为负的处理中也有一些相同的成分。产量和蛋白质含量增量比较显著的处理,其组成的品种间产量和蛋白质含量差异比较大。对两地的30穗重和千粒重,产量与千粒重分析比较表明,混种的增产不是完全通过影响千粒重来实现的。对小麦白粉菌的EST序列进行了SSR位点的搜索和SSR引物的设计,并采用七个采自不同地区的小麦白粉菌株对引物进行了多态性验证,得到7对具多态性的引物对;对这些具有多态性的引物,用小麦条锈,杆锈,和大麦白粉,赤霉菌与小麦白粉菌进行了对比试验,结果如下:在设计出的46条引物对中,有42对扩增出目的条带,有8对具有多态性,其中有7对引物的特异性和多态性较好,可以作为微卫星多态性引物。在具有多态性的引物位点中,有5对引物对应的SSR重复单元为6碱基重复,2对对应的为3碱基重复,1对为2碱基重复。交叉扩增试验表明,开发的小麦白粉菌SSR引物可用于扩增大麦白粉菌、小麦锈菌及赤霉菌。

【Abstract】 Wheat powdery mildew, which is caused by blumeria graminis f.sp. tritici, is a worldwide diseaseoccurring in the major wheat regions. At present main control measures against the expansion of wheatpowdery mildew are breeding for disease resistance and application of chemicals. Use of cultivarmixtures can be a safe, effective and sustainable measure in the control of powdery mildew. In this study,based on the SSR analysis of variety diversity and the resistance of the varieties, we chose eightvarieties. Mixtures with three, four, five, six, or eight varieties and the pure stands were inoculated witha mixture of six powdery mildew isolates and tested for the disease occurrence and some of the yieldcomponents and the content of crude protein in the fields in Langfang, Hebei Province, in2010~2011season. We also chose five varieties based on the above idea, Mixtures with two, three, four and fivevarieties and pure stands were plant in the fields with natural conditions for the disease to occur inXinxiang, Henan Province, in2010~2011. The changes of AUDPC, the disease index, the wheat yieldand the crude protein content were investigated and analyzed. Tthe results are as follows:The analysis of AUDPC for the trial in Langfang shows that the average control efficacy of thethree-cultivar mixtures was9.91%, and the range of control efficacy was-23.23%~45.39%. The averagecontrol efficacy of the four-cultivar mixtures was31.15%, and the range of control was11.19%~56.65%.The average control efficacy of the five-cultivar mixtures was11.53%, and the range of control efficacywas-14.74%~43.67%. The mixtures with four-cultivars are the best in all practices. Comparison of theactual and the theoretical AUDPC in different treatments shows that mixtures with both resistant andsusceptible varieties are better than mixtures with only resistant varieties, and the higher the proportionof the resistant cultivars the better the control efficacy. There is no clear trend that the more diversifiedthe mixtures are the better the control efficacy is.The analysis of DI of Xinxiang used with two methods shows that the average control efficacy ofthe two-cultivar mixtures of the two methods, under flag-leaf investigation and improved “0~9”, were16.07%and4.08%, respectively, and the range of control efficacy was-32.46%~76.42%and-20.85%~44.29%, respectively. The average control efficacies of the three-cultivar mixtures of the twomethods were3.22%and-3.13%, respectively and the ranges of control efficacy were-37.62%~28.78%and-33.33%~24.46%, respectively. The average control efficacy of the four-and five-cultivar mixturesof the two methods were27.75%and12.18%, respectively, and the ranges of control efficacy were5.52%~44.21%and-3.45%~30.02%, respectively. The mixtures with four cultivars are the best in allpractices. Comparison of the actual and the theoretical DI in different treatments shows that mixtureswith both resistant and susceptible varieties are better than mixtures with only resistant varieties, and thehigher the proportion of the resistant cultivars the better the control efficacy. However, there’s no cleartrend that the more diversified the mixtures are the better the control efficacy is.The analyses of wheat thirty-ear weight and crude protein content obtainted in trial in Langfangindicate that the average thirty-ear weight increase of the three-, four-, five-, six-/eight-variety mixtureswere5.07%,4.39%,4.18%and3.24%, respectively. The average crude protein content increase of the three-, four-, five-, six-/eight-variety mixtures were2.57%,3.51%,0.16%and3.44%, respetively. Theanalyses of wheat yield and crude protein content obtained from the trial in Xinxiang indicate that theaverage yield increase of the two-, three-, four-/five-variety mixtures were1.84%,2.96%and2.70%,resptectively. The average crude protein content increase of the two-, three-, four-/five-cultivar mixtureswere0.69%,0.20%and-1.12%, respectively. The mixtures with three and four cultivars are the best inall practices except protein content obtained from trial in Langfang. There were some differences indifferent cultivar mixtures treatments, and there were interactions between varieties composed of themixtures. The mixtures composed with certain varieties displayed more increase to yield while othershad negative impact. For example, Aikang58, Zhengmai004and Zhengnong16had a negative impacton yield;Lunxuan987had a positive impact on crude protein content and Zhengmai9694had anegative impact on Crude Protein Content. The component varieties that have major difference betweentheir yield and Crude Protein Content showed more significant increase in terms of yield and CrudeProtein Content.A number of3815wheat powdery mildew expression sequence tags (ESTs) that originated fromour laboratory were used to search for simple sequence repeats (SSRs). The SSRIT program was runonline and the parameters were set for detection of di-, tri-, tetra-, penta-, and hexa-nucleotide motifswith minimum of6,5,4,3and2repeats, and77simple sequence repeats (EST-SSRs), which accountedfor2.02%of EST database, were identified. Based on these SSR sequences,46EST-SSR primer pairswere designed.42EST-SSR primer pairs can amplify clear bands in7isolates of Blumeria graminis f.sp.tritici, and7primer pairs showed good polymorphisms. Among the7polymophic loci, five are6-baserepeats, two3-base repeats and one two-base repeats. Cross ampliphication indicates that the developedSSR primers can be used to amplify barley mildew, wheat rusts and wheat head blight pathogen.

【关键词】 小麦白粉病AUDPC值病情指数ESTSSR
【Key words】 Blumeria graminis f.sp. triticiAUDPCdisease index (DI)ESTSSR
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