基于TaqMan MGB探针的黑白轮枝菌检测方法

根据黑白轮枝菌(Verticillium albo-atrum)及其近似种β-微管蛋白基因(β-tubulin)序列差异,设计并合成1对引物和1条Taq Man-MGB探针,建立了黑白轮枝菌的实时荧光PCR检测方法。对供试黑白轮枝菌及其近似种实验表明,该方法特异性强,只有黑白轮枝菌可被检出。通过对反应体系的优化,确定了最佳反应条件:引物终浓度为1.0μmol/L,探针终浓度为0.7μmol/L。灵敏度试验结果显示,最低检测限量为总DNA含量10 pg(20μL反应体系)。此方法快速灵敏,为快速检测黑白轮枝菌提供了重要参考。     

棉花内生蜡状芽孢杆菌YUPP-10对棉花黄萎病的防治作用及机制

【目的】棉花黄萎病(Verticillium wilt)是世界性难以防治的病害,筛选有效的生防微生物资源成为解决棉花黄萎病的重要途径之一,本研究旨在分离一株高效拮抗细菌,并明确其防治机理,为生物防治棉花黄萎病提供技术支持。【方法】用葡甘聚糖作为碳源筛选一株能够降解β-1,4糖苷键的棉花内生细菌YUPP-10,通过平板对峙培养、平板对扣培养和悬滴法等测定YUPP-10对大丽轮枝菌(Verticillium dahliae)菌丝生长和孢子萌发的抑制效果;用YUPP-10无菌滤液培养大丽轮枝菌微菌核,检测其对微菌核萌发的影响;利用基质接种法进行盆栽试验探究其对棉花黄萎病的防治效果;通过胚根、叶片接种病原菌,木质化和活性氧爆发研究YUPP-10诱导抗病能力;采用实时荧光定量PCR(qRT-PCR)方法检测防御相关基因的表达。【结果】成功获取一株蜡状芽孢杆菌(Bacillus cereus)YUPP-10,其整体代谢产物在7 d时对大丽轮枝菌的抑菌带宽为0.73 cm,挥发性代谢产物在10 d时对大丽轮枝菌菌落的抑制率为77.03%;YUPP-10无菌培养液对大丽轮枝菌孢子和菌核萌发的抑制率分别为17.22%—71.25%和10.69%—26.62%,且抑制率与无菌滤液的浓度呈正相关。在用YUPP-10玉米蛭石培养物拌土处理的盆栽试验中,其对棉花黄萎病的最高防效达到80.60%。诱导抗性试验发现YUPP-10诱导了胚根和叶片抗大丽轮枝菌的侵染,诱导了细胞木质化和活性氧的爆发。qRT-PCR检测结果显示YUPP-10成功激活了苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、多酚氧化酶(PPO)、几丁质酶(CHI)和病程相关基因(PR10)的表达,对大丽轮枝菌在棉花体内的扩散和生长起到了抑制作用。【结论】YUPP-10通过直接抑制病原菌的生长,同时诱导植物防御反应来抵抗病原菌的侵染和扩展,其具有良好的应用前景。     

Soil suppressiveness and functional diversity of the soil microflora in organic farming systems

Arable fields of 10 organic farms from different locations in The Netherlands were sampled in three subsequent years. The soil samples were analysed for disease suppressiveness against Rhizoctonia solani AG2.2IIIB in sugar beet, Streptomyces scabies in radish and Verticillium longisporum in oilseed rape. In addition, a variety of microbial, chemical and physical soil characteristics were assessed. All data were correlated by multiple regression and multivariate analyses with the objective to find correlations between soil suppressiveness and biotic or abiotic soil characteristics. Significant differences in soil suppressiveness were found between the fields for all three diseases. Multiple regression indicated a significant correlation between suppressiveness against Rhizoctonia and the number of antagonistic Lysobacter spp., as well as with % active fungi and bacterial diversity. Grass-clover stimulated Rhizoctonia suppression as well as the presence of antagonistic Lysobacter spp. (mainly L. antibioticus and L. gummosus) in clay soils. Streptomyces suppression correlated with the number of antagonistic Streptomyces spp., % of active fungi and bacterial population size. The presence of antagonistic Streptomyces spp. correlated with a high fungal/bacterial biomass ratio. Verticillium suppression was only measured in 2004 and 2005, due to the inconsistent suppressiveness along the years. Nevertheless, a significant correlation with pH, potential nitrogen mineralization and bacterial biomass was found. Bacterial and fungal PCR-denaturing gel electrophoresis fingerprinting of bacterial and fungal communities, in general, did not significantly correlate with disease suppression. Highly significant explanatory factors of the composition of the dominating bacterial and fungal populations were % lutum, pH, C/N quotient, biomass and growth rate of bacteria. Additionally, the % of organic matter and years of organic farming were explaining significantly the composition of the bacterial population.Thus, significant correlations between several soil characteristics and suppressiveness of different soil-borne pathogens were found. For two of the three pathogens, suppression correlated with biotic soil characteristics combined with the presence of specific bacterial antagonists. Probably the soil suppressiveness measured in the organic fields is a combined effect of general and specific disease suppression.     

基于PCR检测的4种土传病害病原菌侵染棉花的动态分析

【目的】立枯病、红腐病、枯萎病和黄萎病是新疆棉花上的4种主要土传病害,研究4种病害的病原菌在新疆棉花上的侵染动态,分析各自的侵染始期和最佳防治时期,为病害高效防控提供依据。【方法】针对4种主要病害的病原菌,对混合接菌后分期采集的棉株进行特异性引物PCR检测,依据检测结果,进行各病原菌侵染动态分析及混合侵染分析。【结果】4种病原菌从棉花子叶期即能侵染;立枯丝核菌侵染相对较早、较快,拟轮枝镰孢霉次之;2种病原菌均在棉苗前期有较高的侵染株率,3叶期达到高峰,此后侵染株率逐渐降低;而尖孢镰孢霉和大丽轮枝菌,虽从子叶期开始即可侵染,但侵染株率较低,直到蕾铃期侵染株率呈渐增趋势;病原菌之间的混合侵染普遍存在,以两菌混合侵染居多。【结论】子叶期为4种病原菌的侵染初期;立枯丝核菌和拟轮枝镰孢霉以苗期侵染为特点;尖孢镰孢霉和大丽轮枝菌以子叶期到蕾铃期均可侵染为特点;病原菌之间常有混合侵染发生。     

Development of a Biological Complex Seed-Coating Agent to Control Verticillium Wilt

[Objective] The aim of this study is to make use of the antagonism and growth promoting ability of Bacillus methylophicus strain LH-L3, and add it into the chemical seed-coating agent as an active ingredient. [Method] The optimum formula (agent No.4: 2.5% fludioxonil＋10% thiram＋10% carbofuran＋109 cfu·mL^－1 LH-L3) was obtained based on the sterilization effect of four seed coating agents on infected seed. Then the survival ability of the strain in agent No.4 and its shelf life were identified by plate test. Finally, the disease and pest control effects were tested in the greenhouse pot experiment. [Result] The results showed that the agent No. 4 had the best control effect, and 1.1×105 cfu·mL^－1 of LH-L3 after storage at room temperature for 6 months. Compared with the control, the formula No. 4 increased 11.54% emergence rate, 18.74% plant type height and 20.91% leaf width, respectively, in the 15th day after emergence, and decreased 65.42% and 48.56% of Verticillium wilt at day 2 and day 5 after the disease occurred. The cotton seeds coated with formula No. 4 reduced 83.07% of plant and 92.33% of leaf damaged by Sylepta derogata, compared with the negative control without seed coating. [Conclusion] Strain LH-L3 and chemical pesticide compound as a seed coat agent is completely feasible. And the developed biological complex seed-coating agent has better control effect on Verticillium wilt and Sylepta derogate.     

棉花VIGS病毒载体的构建及其在抗病基因功能鉴定的应用

【目的】利用农杆菌介导的VIGS基因沉默技术,研究陆地棉抗病相关基因在棉花抗黄萎病中的功能。【方法】利用分子生物学技术构建VIGS病毒载体,建立VIGS病毒诱导沉默体系;利用此体系将棉花中的一个钙依赖蛋白(CDPK)基因,通过农杆菌介导在陆地棉中沉默,根据此基因沉默植株在病原菌环境下的发病情况研究该基因在棉花抗病中的功能。【结果】利用包含有GhCPK抗病基因片段的VIGS病毒载体的农杆菌侵染棉花子叶,病毒载体上的目的基因片段由RNA聚合酶识别并合成双链RNA(double stranded RNA,dsRNA),dsRNA由Dicer酶识别并于其它RNA形成RNA诱导的沉默复合体(RNA induced silencing complex, RISC),RISC对内源GhCPK mRNA进行识别和切割作用,内源GhCPK mRNA降解而发生基因沉默。GhCPK基因沉默导致棉苗对黄萎病敏感性增加,证明GhCPK基因参与调控棉花抗病功能。【结论】病毒诱导的基因沉默技术(VIGS)能够快速有效的研究GhCPKs基因在棉花抗病中的功能。     

Comparison of genotyping by sequencing and microsatellite markers for unravelling population structure in the clonal fungus Verticillium dahliae

Microsatellite genotyping of a large sample of isolates of Verticillium dahliae from diverse locations recently identified seven distinct genotypic clusters. However, these clusters were not put in the context of phenotypes known to be correlated with clonal lineages in V. dahliae. The objective of this study was to compare clusters defined by microsatellite markers with clonal lineages defined by single‐nucleotide polymorphisms (SNPs) and vegetative compatibility groups (VCGs). Genotyping isolates known to belong to specific clonal lineages (based on SNPs) with microsatellite markers determined the correspondence of clusters and lineages. All but one cluster corresponded to a known clonal lineage, allowing analysis of correlations of phenotypes with microsatellite genotypes from other studies. As shown previously, most race 1 isolates are in lineage 2A, and most isolates with the defoliating pathotype are in lineage 1A. Phylogenetic incompatibility was used to test for recombination or homoplasy caused by hypervariable microsatellite loci; incompatibility was highly correlated with the number of alleles per locus, suggesting that homoplasy caused by parallel evolution of microsatellite alleles is the cause of incompatibility. Microsatellite genotyping of lineage 1A isolates from cotton and olive in Spain over a 29‐year period revealed remarkably little variation; these markers did not mutate enough to provide insight on the spatial and temporal expansion of this clone. Overall, this study showed that microsatellite genotyping can be used to identify clonal lineages in V. dahliae, which has predictive power for inferring phenotypes of phytopathological relevance such as race and pathotype.     

24种农药与石斛象甲蜡蚧轮枝菌的相容性

测定了8种杀虫剂和16种杀菌剂对石斛象甲蜡蚧轮枝菌菌丝生长的影响。结果表明:杀菌剂对各菌株的抑制性大于杀虫剂,但杀虫剂对菌丝的生长也有不同程度的抑制;杀菌剂中以灰创、霉洁、凯越蓝、乙生、美爽对菌株的抑制性最高,这些杀菌剂不能和蜡蚧轮枝菌混用;杀虫剂中三氟对各菌株的影响最小,可以和蜡蚧轮枝菌同时使用;菌株间对药剂的耐受力区别不大,但不同药剂对菌株的影响相差比较大。     

商丘地区棉花黄萎菌的分离与鉴定

从河南商丘各县区采集棉花黄萎病病株,对其进行黄萎病菌的分离和鉴定。采用连续稀释法和单孢分离法分离得到16个单菌系,并以这些单菌系作为研究对象,利用真菌通用引物ITS1和ITS4对所分离的单菌系内转录间隔区(ITS)进行PCR扩增,得到543 bp大小的片段,经过克隆测序和在GenBank中进行BLASTn分析,与安阳棉花黄萎菌的ITS序列的同源性均达到98%以上,证明了这些片段来自大丽轮枝菌(Verticillium dahliae)的ITS区。同时将这些序列在NCB I的GenBank进行了登记,获得5个登记号:FJ715500、FJ715501、FJ715502、FJ715503、FJ715504。     

棉花黄萎病菌对转hpa1Xoo基因棉花幼苗相对电导率的影响

以两个转hpa1Xoo基因的棉花株系T-33和T-35及其出发品种即非转基因棉品种Z35的2～3叶期棉花幼苗为试材,通过无底塑料盆钵菌液浇根接菌法,在0～10 d不同时段测定叶片浸出液电导率.转基因棉T-34在0～10 d间相对电导率呈增加趋势,10 d时增加率达到115.90%;T-33在接菌后0～1 d间的电导率增幅反而略有下降,但3～10 d的增幅均呈现上升趋势,到10 d时达到140.61%;Z35从接菌后0～10 d的电导率增幅均呈现逐渐上升趋势,到10 d时相对电导率增加率达到180.78%.转基因棉到达相对电导率峰值的时间晚于非转基因棉,其峰值也低于非转基因棉;同时两个转基因棉花株系相对电导率的增加率均低于非转基因棉.被黄萎病菌感染后转基因棉的显症时间较非转基因棉出现晚,两个转基因株系的受危害程度也低于非转基因棉.