拮抗细菌KRS022的鉴定及对大丽轮枝菌的抑制效果

【目的】明确拮抗细菌KRS022的分类地位及对多种病原真菌的抑制作用,重点研究对作物黄萎病的防治效果及其对病原大丽轮枝菌（Verticillium dahliae）的抑制效果,为抗大丽轮枝菌生防制剂的研发提供资源。【方法】通过形态学、生理生化试验及16S rDNA与gyrB基因序列串联分析确定KRS022的分类地位;采用对峙培养法和对扣熏蒸法测试KRS022对多种真菌的抑制作用;采用发酵菌液平板法、涂布法以及对扣熏蒸法明确KRS022对大丽轮枝菌的平板抑制效果;通过无细胞上清共培养法与对扣熏蒸法明确KRS022对大丽轮枝菌孢子萌发及菌丝形态的影响;通过盆栽试验及大丽轮枝菌生物量测定明确KRS022对棉花和烟草黄萎病的防治效果;利用RT-qPCR法检测KRS022激发植物免疫相关基因的表达特性。【结果】拮抗菌株KRS022为革兰氏阴性细菌,鉴定为产碱假单胞菌（Pseudomonas alcaligenes）。该菌株具有解磷、解钾、固氮功能,可产生嗜铁素,具有蛋白酶、淀粉酶、过氧化氢酶活性,属于好氧产碱型细菌,100μg·mL-1氨苄青霉素可作为该菌株的天然抗性筛选条件。KRS022对多种...     

生防菌在棉田土壤中定殖数量与防病作用相关性

【目的】研究拮抗菌施入棉田土壤后生防菌的数量变化和棉花黄萎病的发病情况,分析生防菌的定殖与防病的相互关系,为生防细菌在棉花黄萎病的防治应用提供理论依据。【方法】选择4株棉花内生和根际拮抗菌Bacillus atrophaeus SHZ-24、Bacillus vanillea SMT-24、Bacillu subtilis SHT-15和Bacillus velezensis BHZ-29进行盆栽试验,使用平板计数和实时荧光定量分析细菌和病原菌。【结果】4株拮抗细菌在土壤中数量变化趋势相同,50 d时的存活量从高到低分别为SHZ-24、BHZ-29、SMT-24、SHT-15,4株拮抗细菌处理均可降低棉苗的病情指数。【结论】BHZ-29的防病效果较好。4株拮抗细菌在土壤中的定殖数量与防病效果呈显著正相关。     

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.     

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。     

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.     

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。     

特殊生境中土壤放线菌的筛选与鉴定

采用土壤稀释法分离采自江苏及广西农化厂周边的6份土样,获得50株放线菌。以大丽花轮枝孢、尖镰孢萎蔫专化型和尖镰孢西瓜专化型为靶标菌,通过皿内试验及盆栽试验筛选出5株具有较强拮抗活性的放线菌。菌丝生长和孢子萌发抑制试验表明,菌株JSGF4、JSGF10和GXCK1对靶标菌有较强拮抗作用;盆栽试验表明,5株放线菌对茄子黄萎病有良好防治效果,其中菌株JSGF10对茄子黄萎病的防效高达90.0%;对防效较高的4株放线菌进行形态特征、培养特征及生理生化特性分析,鉴定菌株JSGF4为龟裂链霉菌(Streptomyces rimosus),菌株JSGF10和GXCK1为白色链霉菌(Streptomyces albus),GXDM6为灰色链霉菌(Streptomyces griseus)。     

Fusarium oxysporum Fo47 confers protection to pepper plants against Verticillium dahliae and Phytophthora capsici,and induces the expression of defence genes

The application of the nonpathogenic isolate Fusarium oxysporum 47 (Fo47) reduced the symptoms of verticillium wilt, phytophthora root rot and phytophthora blight in pepper plants. Botrytis cinerea was also tested on the leaves of plants treated with Fo47, but no protection was observed. Verticillium dahliae colonies cultured in the presence of Fo47 grew slower than control cultures, but Phytophthora capsici growth was unaffected by Fo47. At least part of the protection effect observed against V. dahliae could therefore be due to antagonism or competition. In order to search for induced resistance mechanisms, three defence genes previously related to pepper resistance were monitored over time. These genes encode a basic PR‐1 protein (CABPR1), a class II chitinase (CACHI2) and a sesquiterpene cyclase (CASC1) involved in the synthesis of capsidiol, a phytoalexin. These three genes were transiently up‐regulated in the roots by Fo47 in the absence of inoculation with the pathogen, but in the stem only CABPR1 was up‐regulated. In plants that were inoculated with V. dahliae after the Fo47 treatment, the three genes had a higher relative expression level than the control in both the roots and the stem.     

蛇床子提取物对茄子根际土壤中黄萎菌数量及植株抗病性的影响

本试验以茄子根际土壤黄萎菌为作用对象,采用浓度为2、4、8、10 mg/mL的蛇床子提取物,通过田间灌施,配合伤根接菌的方法处理茄子幼苗,探讨蛇床子提取物对根际土壤黄萎菌抑制作用及其对植株抗病性的影响.结果表明,不同浓度蛇床子提取物均可抑制茄子黄萎病的发生,其中浓度为8 mg/mL效果最为显著.与清水对照相比,根际土壤中的黄萎菌数量减少了56.67％～69.23％;茄子植株的发病率和病情指数分别减少了53.12％～54.98％、51.26％～56.82％;生长指标的增幅分别为:株高3.68％～9.59％、茎粗3.41％～9.98％、地上部鲜重4.43％～9.47％、根鲜重2.61％～9.57％、叶面积3.56％～9.55％;防御酶活性也有所增加,增幅分别为:过氧化物酶(POD)45.60％～64.23％、多酚氧化酶(PPO)108.62％～255.00％、苯丙氨酸解氨酶(PAL)62.32％～76.04％.上述结果表明,蛇床子提取物会抑制茄子黄萎病的发生,增加植株的抗病性,促进植株生长.