Development of A Real-Time PCR Assay for Plasmodiophora brassicae and Its Detection in Soil Samples

A SYBR Green I real-time PCR assay was developed to detect and quantify Plasmodiophora brassicae ribosomal DNA(rDNA) and internal transcribed spacer(ITS).A pair of primers PBF1/PBR1 was designed based on the conservative region of rDNA-ITS of P.brassicae.The positive plasmid pB12 was obtained and used as the template to create standard curve.The specificity,sensitivity,and reproducibility of real-time PCR were evaluated respectively.Naturally and artificially infested soil samples containing different concentrations of P.brassicae were detected.The results demonstrated that standard curve established by recombinant plasmid was shown a fine linear relationship between threshold cycle and template concentration.The melting curve was specific with the correlation coefficient of 0.995 and that the amplification efficiency was 93.8%.The detection limit of P.brassicae genomic DNA was approximately 40 copies per 25 μL.The sensitivity of the assay was at least 100-fold higher than conventional PCR.Only DNA from P.brassicae could be amplified and detected using this assay,suggesting the highly specific of this assay.The coefficient of variation was less than 3%,indicating the PCR method revealed high reproducibility.The detection limit in soil samples corresponded to 1 000 resting spores g-1soil.Bait plants were used to validate the real-time PCR assay.This developed real-time PCR assay allows for fast and sensitive detection of P.brassicae in soil and should be useful in disease management and pest interception so as to prevent further spread of P.brassicae.     

大白菜根肿病菌生理小种鉴定研究

为明确我国各地根肿病菌致病性的不同，分别使用Williams 鉴别系统和具有不同根肿病抗性的4 个大白菜品种对采 自全国多地的20 份大白菜根肿病菌菌源进行生理小种和致病型的划分。利用Williams 鉴别系统进行鉴定，其中18 份为4 号 生理小种，2 份为2 号生理小种。利用4 个大白菜品种进行鉴定，根据致病力不同划分为6 种类型，其中4 号生理小种存在 6 种类型，2 号生理小种存在2 种类型。认为Williams 鉴别系统已不能精确划分大白菜根肿病菌的生理小种，而研究筛选具 有不同抗病类型的大白菜品种（品系）鉴定划分根肿病菌致病型具有重要意义。     

十字花科根肿病的研究进展

概述了根肿病的危害与发生、病原菌的基本特征、病原菌的分离提纯与人工接种方法以及十字花科大白菜抗根肿病的研究等。     

芸薹根肿菌毒素对油菜幼苗生理 性状及生长的影响

为探索芸薹根肿菌的致病作用、采用水培法研究根肿菌毒素对油菜胚根和胚芽生长尧幼苗致萎性尧细胞 膜透性尧叶绿素含量尧根系活力和幼苗生长的影响。结果表明院根肿菌毒素具有明显的生物活性、能够抑制油菜胚根 和胚芽的生长、且随处理时间的延长和处理浓度的增加、幼苗萎蔫级数逐渐增加。毒素的作用可使油菜细胞膜透性 增加、叶绿素含量和根系活力下降、其中叶绿素b 的含量随处理时间的延长表现为先上升后下降的趋势、但仍低于 对照。油菜幼苗的株高和主根长随毒素浓度的提高均呈逐渐下降的趋势、在85%和100%毒素浓度胁迫下、油菜幼苗 株高及主根长均显著低于对照、说明植株的生长受到明显抑制。     

大白菜NPR家族基因鉴定及表达模式分析

为明确大白菜NPR转录辅助因子在大白菜基因组中的数量、结构和表达特点,揭示NPR在水杨酸信号途径调控大白菜生长发育过程中的作用。本研究利用拟南芥资源信息库TAIR10和芸薹属基因组网站Brassica Database鉴定大白菜基因组中的NPR家族成员,并对其蛋白和基因特性进行生物信息学分析。鉴定出大白菜基因组中包括11个NPR家族基因,分别定位在大白菜7条染色体上,具有1~5个外显子不等。BrNPR家族蛋白可聚类为6个分支,包含11个保守元件,并且所有BrNPR家族成员均含有DUF和Ank两个保守结构域,其中BrNPR1-4成员基因的编码区均含有核定位信号(NLS)。基因表达模式检测结果表明部分大白菜NPR基因在不同组织中具有组织特异的表达模式,BrNPR1B在根、茎和叶及BrNPR3B在花和种荚中表达显著,绝大部分BrNPR基因受盐胁迫诱导表达,部分BrNPR基因受根肿菌侵染诱导表达。本研究可为后续大白菜NPR基因的功能验证提供依据。     

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.)

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. ‘Akiriso’, a clubroot-resistant F₁ cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in ‘Akiriso’. CR in ‘Akiriso’ segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar ‘CR Shinki’. We developed additional markers around CRb and constructed partial genetic maps of this region in ‘Akiriso’ and ‘CR Shinki’. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F₂ populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in ‘Akiriso’ is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.     

Transfer of resistance to race 2 of Plasmodiophora Brassicae from Brassica napus to cabbage (B. oleracea var. Capitata). I. Interspecific hybridization between B. napus and B. oleracea var. Capitata

Summary Interspecific hybridization between Brassica napus L. (2n=38, a₁a₁c₁c₁) and B. oleracea var. capitata L. (2x- and 4x-cabbage; 2n=2x=18, cc and 2n=4x=36, cccc) was carried out for the purpose of transferring clubroot disease resistance from the amphidiploid species to cabbage. Nineteen hybrids with three different chromosome levels (2n=28, a₁c₁c; 2n=37, a₁c₁cc and 2n=55, a₁c₁cccc) were obtained. The F₁ plants were mostly intermediate between the two parents but as the number of c genomes in the hybrids increased, the more closely the hybrids resembled the cabbage parent. All F₁ hybrids were resistant when tested against race 2 of Plasmodiophora brassicae wor. The complete dominance of resistance over susceptibility suggested that the gene(s) controlling resistance to this particular race of the clubroot pathogen is probably located on a chromosome of the a genome in Brassica.Contribution No. J654.     

枯草芽孢杆菌XF-1提取物对11种植物病原菌的抑制作用

采用盐酸和甲醇抽提法,提取了枯草芽孢杆菌XF-1(Bacillus subtilis XF-1)的抑菌物质。以该提取物处理根肿菌孢子2 d,有50%孢子活性被抑制;处理9 d后,孢子萌发相对抑制率始终保持在90%以上。提取物对三七根腐病菌、水稻纹枯病菌、禾谷镰刀菌、魔芋基腐菌、烟草赤星菌生长抑制率分别为71.97%、62.23%、60.96%、60.70%和57.92%。     

甘蓝根肿病接种方法的研究

分别比较了菌土接种、蘸根接种对发病的影响,结果显示菌土接种的发病最严重;在接种浓度试验中,浓度越高,发病越重;对不同的根肿菌进行接种比较发现重庆涪陵地区的根肿菌比成都地区根肿菌致病性强,以涪陵地区的根肿菌作为菌源,验证了2个抗病材料99033,99037和2个感病材料99076,99046。并根据实践建立了一套新的田间单株病情分级标准。     

土壤中芸薹根肿菌qPCR检测与风险预警体系的建立与应用

 建立了土壤中芸薹根肿菌荧光定量PCR(qPCR)快速检测及风险预警体系。确定了芸薹根肿菌qPCR检测的特异性引物PbF/PbR,对根肿菌质粒DNA的检测灵敏度为1.612×10^-6 ng·μL^-1,比普通PCR高出1 000倍;对土壤和基质中芸薹根肿菌孢子的最低检测下限均为10 个·g^-1,而土壤和基质带菌的发病阈值分别为100和1 000 个·g^-1,高于该浓度时根肿病发生风险大。本研究建立的芸薹根肿菌qPCR技术体系检测下限远低于发病阈值,可以快速、准确、定量地检测出采自四川绵阳、湖北恩施、江苏无锡、山东青岛、辽宁沈阳、山西运城、内蒙古巴彦淖尔和宁夏固原等8个地区的27份田间土壤中芸薹根肿菌的数量,实现对十字花科根肿病的监测预警,为制定产前病害防控方案提供依据。