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。     

Influence of cultivar resistance and inoculum density on root hair infection of canola (Brassica napus) by Plasmodiophora brassicae

The impact of cultivar resistance and inoculum density on the incidence of primary infection of canola root hairs by Plasmodiophora brassicae, the causal agent of clubroot, was assessed by microscopy. The incidence of root hair infection in both a resistant and a susceptible cultivar increased with increasing inoculum density, but was two‐ to threefold higher in the susceptible cultivar; the relationship between root hair infection and inoculum density was also substantially stronger and more consistent in the susceptible cultivar. In the susceptible cultivar, the root hair infection rate peaked between 6 and 8 days after sowing and then declined. In the resistant cultivar, it increased over the 14‐day duration of each study. It appears that examination of root hair infection by microscopy in a bait crop of susceptible canola could serve as a useful tool for estimating P. brassicae inoculum levels in soil. In a separate trial, the relationship between inoculum density and clubroot severity, plant growth parameters, and seed yield was assessed under greenhouse conditions. Inoculum density in the susceptible genotype was strongly and positively correlated with clubroot severity and negatively correlated with plant height and seed yield. In addition, a single cropping cycle of the susceptible cultivar contributed significantly higher levels of resting spores to the soil in a greenhouse test than did a cycle of the resistant cultivar, as assessed by quantitative PCR and microscope analysis.     

应用实时荧光定量PCR技术检测土壤中根肿病菌休眠孢子

为准确、快速、便捷检测出土壤中根肿病菌休眠孢子量,应用所建立的根肿病菌实时荧光定量聚合酶链式反应检测技术,测定了湖南湘潭县响水乡、长沙县路口镇、桃江县桃花江镇十字花科作物根肿病发生地的42份土样的休眠孢子数量。检测结果表明,40份土样的根肿病菌休眠孢子量为(2.76×104~4.37×106)个/g;休眠孢子量≥104个/g的土壤均有根肿病发生,而休眠孢子量为8.42×102、9.78×102个/g的2份土样未发生根肿病,说明当土壤中根肿病菌休眠孢子量≥104个/g时,根肿病害易发生。     

不同大白菜品种对根肿病的抗性鉴定

采用Willimas 鉴别系统对采自云南省安宁、玉溪地区的2 份根肿病菌菌样进行鉴定。结果表明：4 个鉴别寄主均为阳性,说明这2 份菌样均为根肿病菌4 号生理小种。采用菌土置入法对从国内外引进的18 个抗根肿病大白菜品种进行抗病性鉴定。结果表明：CR-惠民、康根51 和泰能舒根对该生理小种均具有抗性,发病率分别为0、0 和4%,可利用其作为抗源转育抗病新材料。对CR-惠民和康根51 的田间调查结果表明,这2 个品种的经济性状优良,可以作为云南省安宁、玉溪地区大白菜抗根肿病育种的抗源材料。     

十字花科蔬菜品种对根肿菌4号和9号小种的抗性

依据江西省内根肿病菌的生理小种类群分布,分别在婺源县4号小种区域和南昌县9号小种区域设立田间病圃,于2016年9—12月对108个十字花科蔬菜(花椰菜、包菜、萝卜、大白菜、小白菜)品种进行抗性鉴定,并随机选取其中29个品种进行人工接种鉴定。结果表明:十字花科不同种间蔬菜对根肿菌不同小种的抗性存在差异,种内品种对同一小种的抗性也存在差异;花椰菜、包菜和萝卜对9号小种均有抗性,上海黑叶五月慢、新奶白、京绿乌塌菜3个小白菜品种和京夏王、北京桔红心、京春娃2号等13个大白菜品种对9号小种有抗性;参试品种对4号小种的抗性不如对9号小种的,但仍筛选到紧花6、紧花16、松花2等10个花椰菜品种和满堂红萝卜品种可在4号小种区域种植。     

Flooding affects the development of Plasmodiophora brassicae in Arabidopsis roots during the secondary phase of infection

Clubroot, a disease of Brassicaceae species, is caused by the soilborne pathogen Plasmodiophora brassicae. High soil water content was previously described to favour the motility of zoospores and their penetration into root cells. In this study, the effect of irrigation regimes on clubroot development during the post‐invasive secondary phase of infection was investigated. Three irrigation regimes (low, standard, high) were tested on two Arabidopsis accessions, Col‐0 (susceptible) and Bur‐0, a partially resistant line. In Col‐0, clubroot symptoms and resting spore content were higher under the ‘low irrigation’ regime than the other two regimes, thus enhancing the phenotypic contrast between the two Arabidopsis accessions. Clubroot severity under high and low irrigation regimes was evaluated in near‐isogenic lines derived from a Col‐0 ×  Bur‐0 cross, to assess the effect of soil moisture on the expression of each of four quantitative trait loci (QTL) controlling partial resistance. The presence of the Bur‐0 allele at the QTL PbAt5.2 resulted in reduced severity only under low irrigation, whereas the Bur‐0 allele at QTL PbAt5.1 was associated with partial resistance only under high irrigation. QTL PbAt4 reduced the number of resting spores in infected roots, but was not associated with reduced clubroot symptoms. The results indicated that soil moisture could have consequences for the secondary phase of clubroot development, depending on plant genotype. Future genetic studies may benefit from using combinations of watering conditions during the secondary stage of infection, thus opening up the possibility of identifying genetic factors expressed under specific environmental conditions.     

氟唑活化酯诱导大白菜抗根肿病的研究

本文研究了我国具有自主知识产权的新型化合物氟唑活化酯诱导大白菜对根肿病的抗病效果及诱导技术。荧光染色观察发现该化合物对根肿菌休眠孢子没有明显的杀死作用,但具有明显的诱导抗病效果。不同的诱导浓度、诱导间隔期和诱导次数对根肿病的诱抗效果均不同。温室盆栽试验结果显示,25 mg/L的氟唑活化酯间隔7 d诱导大白菜3次,对根肿病的防病效果最好,达到71.33%,与诱抗剂苯并噻二唑效果相当,显著高于杀菌药剂50%氟啶胺悬浮剂拌土的防效(63.32%)。田间从苗期开始用氟唑活化酯25 mg/L诱导大白菜3次,诱导间隔期为7 d,对根肿病的诱抗效果最好达69.99%±2.01%,略低于对照杀菌剂氟啶胺拌土施药。本文明确了新型诱抗剂氟唑活化酯诱导大白菜对根肿病的抗病效果,并系统研究了施药技术,为白菜根肿病的防治提供了新手段。     

芸薹根肿菌次生游动孢子侵染致病分析

为明确芸薹根肿菌Plasmodiophora brassicae Woron.在其它寄主中是否广泛存在无性短循环及次生游动孢子的侵染致病性,以不结球白菜为寄主培养3批幼苗(G1、G2和G3),用休眠孢子悬浮液接种G1,被侵染的G1接种G2,被侵染的G2接种G3,采用离心管水培法研究其侵染致病性。结果显示,无性短循环研究中,G1、G2和G3根毛均被侵染,除G3并株接种侵染率为33.33%外,其它处理侵染率均在50.00%以上,根毛里有明显的游动孢子囊;次生游动孢子能侵染不结球白菜的皮层组织,致使不结球白菜发病形成明显的肿根;G1、G2和G3水培发病率为20.00%、15.00%和6.00%,砂培发病率为22.50%、18.75%和7.50%;G3肿根病理切片中可观察到休眠孢子。表明芸薹根肿菌侵染不结球白菜时,其生活史中存在无性短循环,次生游动孢子具有侵染致病作用。     

Histological methods to detect the clubroot pathogen Plasmodiophora brassicae during its complex life cycle

The clubroot pathogen Plasmodiophora brassicae is an obligate biotrophic protist that lives in close relationship with its host cell. The roots of the host plants are colonized and the plant growth is altered upon infection. While shoots can be stunted and show wilt symptoms after longer infection periods, the root system is converted to a tumorous root tissue, called ‘clubroot’, by alterations of the plant growth promoting hormones auxin, cytokinin and brassinosteroid. Because the life cycle occurs largely within the host cells, this leads to dramatic changes in host root morphology and anatomy. Thus, the identification of the respective protist structures in the host tissue by microscopy is challenging. Different staining methods as well as fluorescence and electron microscopy of thin sections can reveal specific life stages of P. brassicae and can yield additional information on the changes in the host tissues concerning, for example, cell wall properties. In addition, promoter–reporter fusions, immunostaining methods and in situ hybridization techniques can be used to gain additional information on the changes in the host roots.