【作者】 李六英；

【导师】 马冠华；

【作者基本信息】 西南大学 ， 植物病理学， 2018， 硕士

【摘要】 烟草赤星病是发生于烟草成熟后期的叶部真菌病害,严重威胁着世界各产烟区的烟叶产量和质量。给我国国民经济也造成了严重的损失。生产上主要以化学药剂防治为主,杀菌剂的长期大量使用,烟草赤星病的抗药性问题日益突出。了解烟草赤星病的病原菌组成和病原菌的遗传基础,可促进对烟草赤星病害的全面认识,为制定有效的赤星病防治措施提供信息。本研究主要对烟草赤星病菌的分离鉴定、遗传多样性、致病力分化以及碳氮源利用进行了系统地分析,主要研究结果如下:1.2014²016年间于我国重庆等9个省市的烟草生产区采集典型的烟草赤星病样品（300份）,通过单孢分离共获得289株赤星病菌。从各省市随机挑选15株共135株进行了系统的形态学鉴定,发现其中有70株链格孢菌Alternaria alternata和65株长柄链格孢菌A.longipes;采用引物ITS1和ITS4对135株病原菌进行PCR扩增,并构建系统发育树,供试菌株均与NCBI下载的小孢子种链格孢聚为一组,而与大孢子种链格孢各聚为一组,表明供试菌株为链格孢属小孢子种,ITS序列可作为鉴定烟草赤星病菌的辅助技术。2.采用正交试验设计方法对影响ISSR-PCR反应的主要因子Mg²⁺,dNTP,Taq DNA聚合酶和引物4个因素进行了优化试验,并进行梯度PCR试验确定最适退火温度和循环次数,且验证稳定性,建立了赤星病菌的ISSR-PCR最佳反应体系。在25μL总反应体系中含1.00 mmol/L Mg²⁺,0.15 mmol/L dNTP,0.30μmol/L引物,0.75 U Taq DNA聚合酶,1×PCR Buffer和30.00 ng DAN模板。扩增程序为94℃预变性4 min,94℃变性1 min,50℃退火1 min,72℃延伸90 s,共36个循环,最后72℃延伸10 min。从40条初筛引物中,筛选出扩增条带清晰、多态性及稳定性较好的17条ISSR引物。3.17条引物分别对135株烟草赤星病菌的基因组DNA进行ISSR-PCR扩增,共扩增出192条谱带,其中多态性谱带177条,多态率为92.19%,扩增片段的大小介于100³000 bp。UPGMA聚类分析显示,链格孢菌和长柄链格孢菌的遗传相似性系数分别在0.67¹.00和0.66¹.00之间,遗传相似系数为0.83可使链格孢菌和长柄链格孢菌分别划分为5个和6个亚群,其中链格孢菌的不同地理种群间表现出一定的地理相关性,长柄链格孢菌的不同菌株随机分组。PopGene分析显示,病菌种群的基因多态性和遗传多样性丰富,链格孢菌和长柄链格孢菌的群体间遗传分化系数(G_ST)分别为0.36和0.37,均存在遗传分化,两种赤星病菌的遗传分化结构表现出相似性;群居每代迁移数（Nm）分别为0.89和0.85,不同地理种群间存在基因交流。表明链格孢菌和长柄链格孢菌均存在丰富的遗传多样性,且二者进化方向相似,ISSR标记能较好地揭示烟草赤星病菌种群之间的亲缘关系和遗传差异性。4.采用菌丝块创伤接种法,测定了135株赤星病菌对云烟87离体叶片的致病力,链格孢菌和长柄链格孢菌的致病力均可分为强、较强、中等、弱和无致病力5个类型,都以致病力较强和致病力中等两个类型的分布比例较高,致病力强、致病力弱和无致病力分布较少且均衡。两种病原菌,其中致病力较强类型的最高分布比率出现在链格孢菌中,占38.6%,而致病力强类型的分布比例最高是长柄链格孢菌,占10.8%。不同省市的菌株在5个致病力类型中分布差异较大,表明我国烟草赤星病菌致病力分化差异明显。5.采用全自动微生物鉴定系统分析2株链格孢菌CQ1（中等致病力）和GZ11（中等致病力）及2株长柄链格孢菌HuN2（强致病力）和YN4（弱致病力）对PM1、PM2中190种碳源物质以及PM3中95种氮源物质的利用情况,结果显示,4株赤星病菌对3个微孔板中碳氮源物质利用总体一致,碳源利用率约为24.21%,氮源利用率约86.31%。不同致病力菌株的利用也表现出差异,长柄链格孢菌的强致病力菌株HuN2比弱致病力菌株YN6对D-甘露糖、D-木糖、D-甘露醇、b-环糊精L-缬氨酸、D-天门冬酰胺、腺苷和i-赤藓糖醇等物质利用更好;链格孢菌的中等致病力菌株CQ1比GZ11对L-鼠李糖、海带多糖和二羟基丙酮利用更好;且弱致病力菌株YN4对水杨苷的利用比强致病力菌株HuN2和中等致病力菌株CQ1、GZ11都好。表明烟草赤星病菌种群内个体间对碳氮源物质的利用上存在差异,从表型上证实我国烟草赤星病菌丰富的遗传多样性。更多还原

【Abstract】 Brown spot,one of the most destructive leafspot diseases on tobacco,seriously threaten the tobacco yield and quality worldwide.It also caused serious losses to national economy in China.At present,the chemical control still plays an important role in the disease management,however,the applications of the fungicides over a large geographic scale can induce the emergence of resistant strains in the pathogen population.Learning about the composition and the genetic structure of the pathogen of tobacco brown spot,is helpful for the consideration of the disease effective controls,for this purpose,the pathogen identification,pathogenicity,genetic diversity,and the carbon and nitrogen utilization of the pathogens were studied.The results showed.1.Specimens with typical symptoms of tobacco brown spot were collected from 9provinces or cities such as Chongqing from 2014 to 2016（300 samples）,and totally 289strains were obtained.Then 135 strains were selected in random for further studies.Morphological identification showed 70 of them were Alternaria alternate and 65 were A.longipes.rDNA-ITS molecular analysis of 135 strains was carried out,the phylogenetic tree suggested that all sequences of tested strains gathered with downloaded microspores and wholely separated from megaspores downloaded.Therefore,135 strains consequently were proved to be microspores of Alternaria and ITS sequence could be used as a assistive technology for pathogen identification of tobacco brown spot.2.Orthogonal design was applied to optimize the ISSR-PCR reaction system for A.alternata and A.longipes of tobacco brown spot.The concentration of Mg²⁺,dNTP,Taq DNA polymerase and primers that affecting the quality of amplification mainly were tested respectively,and the annealing temperature and cycle times were proposed by gradient PCR.The results showed that the 25μL total reaction volume contained 1.00mmol/L Mg²⁺,0.15 mmol/L dNTP,0.30μmol/L primers,0.75 U Taq DNA polymerase,1×PCR Buffer and 30.00 ng template DNA.Amplification procedure by PCR was pre-denaturation at 94℃for 4 min,36 cycles of denaturation at 94℃for 1 min,annealing at 50℃for 1 min and extension at 72℃for 90s,then a final extension at72℃for 10 min.The new established reaction system and procedure were stable and reliable tested by amplification of 24 strains of Alternaria using primer UBC841.17ISSR primers3.The genetic diversity analysis of 135 strains were carried out by using inter-simple sequence repeats.The results showed that a total of 192 bands were amplified by 17 ISSR primers,of which 177 were polymorphic,and the polymorphism percentage was 92.19%,the amplification fragments ranged from 100³000 bp.Cluster tree was formed using UPGMA method,consequently the similarity coefficients of A.alternata and A.longipes respectively ranged from 0.67-1.00,and 0.66-1.00,at the similar level of 0.83,A.alternata and A.longipes tested were severally divided into 5and 6 subgroups,among which the different geographic populations of A.alternata appeared geographical correlation,but A.longipes didn’t.The PopGene analysis results revealed that the genetic diversity and genetic differentiation of A.alternata and A.longipes groups in China were abundant,and the coefficient of population genetic differentiation(G_ST)respectively was 0.36 and 0.37.The number of migrants per generation（Nm）respectively was 0.89 and 0.85,which indicated that the genetic information exchange was frequent among A.alternata and A.longipes in different geographic regions.Population genetic structure of two types of pathogens appeared to be similar.The present study showed that both A.alternata and A.longipes existed abundant genetic diversity which naturely had the similarity of evolution direction,ISSR markers could be used to analyze the genetic relationship and genetic diversity of pathogens of tobacco brown spot.4.Inoculation method of mycelium block was used to test the pathogenicity of 135strains in vitro leaves of Yunyan 87.The results showed that both A.alternata and A.longipes strains could be divided into five types,most virulent,relatively powerful virulent,moderately virulent,weak virulent and avirulent strains.Virulence distributions of strains between A.alternata and A.longipes were similar obviously.Avirulent strains were found in both species,the occurrence of relatively powerful virulent and moderately virulent strains were higher,but most virulent strains as well as weak virulence lower.The highest distribution ratio of relatively powerful virulent strains were found in A.alternata,the rate of them was 38.6%,and A.longipes owned the highest incidence of most virulent strains,the rate was 10.8%.What’s more,there were obvious difference in five types of strins from different provinces or cities.That’s indicated there were evident differentiation among the pathogens of tobacco brown spot in China.5.CQ1（moderately virulent）and GZ11（moderately virulent）of A.alternata as well as HuN2（most virulent）and YN4（weak virulent）of A.longipes were selected to use automatic microbial identification system to analyze the utilization about 190 kinds of carbon source materials of PM1 and PM2,95 kinds of nitrogen source of PM3.The results revealed that the utilization of carbon and nitrogen sources of 4 tested fungi strains were roughly similar,the utilization ratio of carbon and nitrogen sources were24.21%and 86.31%respectively.There were also some differences among them,for A.alternata,strain HuN2 with most virulent than weak virulent YN6 had a better use of D-Mannose,D-Xylose,D-Mannitol,b-Cyclodextrin,I-Erythritol,L-Valine,D-Asparagine,Adenosine,and so on.For A.alternate,moderately virulent CQ1 had a better ability in use of L-Rhamnose,Laminarin and Dihydroxyacetone than GZ11.Additionally,the weak pathogenic strain YN4 was better than HuN2,CQ1 and GZ11 in the use of Salicin.That’s indicated there were obvious ulitization variance in different types of pathogenicity of strains,which confirmed that the genetic diversity of pathogen of tobacco brown spot among different populations was abundant in China.更多还原