芸薹根肿菌排孢管的显微与超显微结构观察

正根肿病是重要的十字花科作物土传病害,病原菌为专性寄生的芸苔根肿菌Plasmodiophora brassica Woron.,属根肿菌目芸苔根肿菌属,主要侵染寄主植物根部形成根肿症状。根肿菌侵染包括根毛侵染和皮层中柱侵染2个阶段,在外界环境条件适宜的情况下,根肿菌休眠孢子萌发释放的初生游动孢子侵染寄主根毛并形成游动孢子囊产生次生游动孢子,次生游动孢子再侵染根毛或者是成对融合进而     

芸薹根肿菌（Plasmodiophora brassicae）单孢分离接种及生理小种的鉴定

芸薹根肿菌(Plasmodiophora brassicae)单孢分离接种方法曾有报道,本试验改进了国外单孢分离技术.将休眠孢子悬浮液稀释到1个/0.5 μL,滴在一块消过毒的载玻片上,放置在显微镜下反复检查,以确保它精确只含有一个孢子.用微管将孢子吸入接种在2日龄白菜幼苗根毛上,营养液培养3周.为使其更接近田间发病环境条件,后期将疑似发病根在无菌土中继续进行病菌增繁.该方法能够有效地加快病菌繁殖感染,提高单孢分离接种成功率.对不同地区病菌进行大量单孢分离接种.本试验对3个地区的病菌单孢接种共950株,接种成功27株,使用Williams根肿病菌鉴别寄主鉴定,共9个生理小种.     

土壤pH对芸薹根肿菌侵染及病害发生的影响

通过设置不同pH梯度,研究土壤pH对根肿菌侵染及病害发生的影响。结果表明:土壤酸性时病菌侵染速度快,碱性时慢,而强酸性和碱性土壤条件则抑制孢子萌发;pH为6.0时最有利于根肿菌休眠孢子萌发,萌发率最高,为53.96%;碱性条件可使初级原生质团变形凝结成球状,不能正常分裂或延迟形成游动孢子囊,从而不利于根肿菌侵染。白菜发病率与病情指数随pH升高,呈先上升后下降趋势。其中,pH为5.0时,发病率和病情指数最高,pH 7.0~8.0时发病轻。因此,适宜的偏酸性环境条件下,通过作用于病菌休眠孢子萌发和侵染,提高病害危害程度,而中性或碱性条件干扰该过程并降低病害发生。     

基于根肿菌早期侵染量的白菜抗性分析

为探究不同抗性白菜品种对根肿菌侵染量的影响,通过水培法观测4个白菜品种接种根肿菌后其在寄主根内的发展动态,实时荧光定量PCR检测技术对同期根内含菌量进行分析,并结合温室盆栽试验获得的各品种发病数据进行相关性分析。结果表明,早熟长江5号根肿病的发病率和病情指数分别为82.5%和36.2,华良早五号为66.7%和37.5,CR-春美为68.9%和36.0,可归为感病品种,而CR-英雄为52.5%和16.5,可归为抗病品种;水培法接种观测和PCR定量结果显示,根毛侵染率、皮层侵染数以及根内含菌量均表现为CR-英雄最低,显著低于其它3个品种。根毛侵染率、皮层侵染数、根内含菌量、发病率四者间呈显著相关,其R2最大值变幅为0.84~0.98,且根毛侵染率与根内含菌量、发病率与根内含菌量的最大相关系数均出现在第6天,说明根肿菌早期侵染量直接影响白菜根肿病的发生程度。表明根内早期侵染是根肿菌致病的关键环节,通过水培法观测并结合实时荧光定量PCR检测可以评价品种的抗性,可作为根肿病抗性评价的一种方法。     

实时荧光定量PCR技术在湖南油菜根肿病菌检测中的应用

为完善湖南油菜根肿病的监测预警体系, 分析油菜根肿病的发生与芸薹根肿菌休眠孢子量的关系, 防止湖南油菜根肿病进一步蔓延?本研究依据芸薹根肿菌ITS序列设计了一对特异性引物, 通过制备标准质粒构建了快速?精准的实时荧光定量PCR检测方法?其检测灵敏度为3×10-10 μg/μL, 比常规PCR 高100 倍; 利用建立的方法检测了来自湖南各地土壤样品中芸薹根肿菌的休眠孢子数量, 64份土样中的休眠孢子含量基本大于104个/g, 所检测的土样中孢子含量最高为2.22×107个/g?据实地调查, 当土壤中休眠孢子数量大于 104个/g 时, 油菜发生根肿病的风险较大, 休眠孢子含量较高的田块其发病程度也较为严重?结果表明, 建立的实时荧光定量PCR检测方法能对油菜根肿病进行早期诊断, 为湖南油菜根肿病发生的监测预警提供有效技术参考?     

根肿病菌侵染时期与浓度对寄主生长特性及抗性的影响

[目的]为探讨根肿病菌接种浓度与时期对寄主发病的交互作用及根肿病菌侵染后寄主的抗性响应.[方法]通过苗期盆栽试验,系统地研究了接种根肿病菌浓度(105、106、107、108 cfu/mL)及时期(0、10、20 d)对白菜发病率及防御酶活性的影响.[结果]发病率及病情指数与接种根肿病菌浓度呈正相关,108 cfu/mL为最佳侵染浓度,白菜幼苗出现第一片真叶后10 d接种侵染效果最佳.根肿病侵染后,根肿病发病程度与呼吸速率和气孔导度呈正相关,根系活力随病情指数先升高后降低,植株受根肿病侵染后光合作用降低,呼吸作用增强,植物生长受抑制;防御酶POD、PAL、PPO与发病率呈负相关,丙二醛含量与发病率呈正相关,植株抵抗病害能力降低.[结论]研究为根肿病的侵染和防控提供参考.     

江西省蔬菜根肿病菌致病性分化研究

正芸薹根肿病菌(Plasmodiophora brassicae)存在生理小种分化现象,国际上通常采用Williams鉴别系统~([1])和欧洲根肿病菌鉴别寄主(ECD)系统~([2])鉴定根肿病菌生理小种。我国尚没有研究出适合本国根肿病菌生理小种鉴定的鉴别寄主系统。国内学者研究根肿病菌生理小种通常采用Williams鉴别系统~([3])。为了明确江西省蔬菜根肿病菌致病性分化及病菌小种的变化趋势,作者采用     

大豆疫霉根腐病菌单游动孢子的毒性遗传与变异

 采用离体叶柄伤口接种法测定大豆疫霉根腐病菌44号生理小种单游动孢子连续3代或4代分离后代的毒性,结果表明:从S1中选择与亲本相比毒性不发生变异的1号单游动孢子菌株(44号生理小种)和变异最大的30号单游动孢子菌株(1号生理小种)继续分离2代或3代,单游动孢子毒性变异趋势主要是从44号生理小种变异为3号生理小种,也有变异成毒性公式为7或1a,7的单游动孢子。大豆疫霉根腐病菌无性世代毒性变异几率很高,多数单游动孢子毒性在分离后代中都发生变异,产生不同的小种或毒性公式,并且毒性变异基本不能稳定遗传。     

蚕豆油壶菌疱疱病及其防治的初步研究

 蚕豆疱疱病是川西北高原春蚕豆的主要病害之一,发病面积约6万亩。罹病茎、叶起疱,一般因病减产20%左右。病原为巢豆油壶菌(Olpidium viciae)。巢豆油壶菌及其所致病害发生于日本,我国尚属初次发现。该菌寄主范围广,易见的自然寄主为蚕豆、豌豆和歪头菜。在人工接种下,除可侵染大豆、菜豆、箭舌豌豆、兵豆等多种豆科作物外,也能侵染油菜、结球甘蓝、大白菜、萝卜、黄瓜、南瓜、莴苣、菠菜、荞麦等作物。以病株残体内的休眠孢子囊在土内越冬,翌春萌发侵入寄主,在寄主细胞内形成游动孢子囊,通过出管释放单鞭毛游动孢子进行再侵染,潜育期13~18天。游动孢子亦起配子作用,结合形成双鞭毛游动合子,以合子侵入。后期在寄主细胞内形成休眠孢子囊。     

青花菜根肿病苗期抗性鉴定技术的建立

为建立快速、有效的青花菜根肿病苗期抗性鉴定技术,将芸薹根肿菌Plamodiophora brassicae Woron.人工接种于高感根肿病青花菜自交系90196,研究了接种菌液浓度、接种寄主苗龄、接种基质p H和接种方法等对人工接种鉴定效果的影响。结果表明,在一定范围内,根肿病发病率及病情指数随着接种菌液浓度的升高而增大,接种菌液浓度为3×108CFU/m L时,发病率及病情指数分别为97.22%和86.11,可以反映寄主真实的抗性水平;接种寄主苗龄为2~6叶期均能使植株发病,但2~3叶期发病效果最佳;接种基质偏酸性(p H 5~6)有利于根肿病的发生;使用伤根灌菌法进行鉴定,青花菜根肿病发病率和病情指数均最高,分别为100.00%和88.10,优于蘸根法和浸芽法。用已知抗性水平的12个自交系和8个杂交种进行验证,鉴定结果表明该苗期抗性鉴定技术可客观反映供试材料的实际抗性水平。     

三种寄主植物对小菜蛾实验种群特征的影响

为明确萝卜、白菜和油菜对小菜蛾实验种群特征的影响,测定了小菜蛾对3种寄主的取食量,通过构建两性生命表比较了不同寄主植物对其实验种群发育与繁殖的影响,并综合生命表与取食量参数,评价了小菜蛾对萝卜、白菜和油菜的危害。结果显示:小菜蛾幼虫在萝卜、油菜和白菜上的取食量差异显著,分别为93.3、105.0和114.7 mg/头。小菜蛾取食萝卜、白菜和油菜后,其幼虫的发育历期差异显著,依次为8.6、8.5和7.6 d;种群内禀增长率依次为0.18、0.23和0.23,周限增长率为1.19、1.26和1.26,净生殖率为18.7、46.1和30.6。取食萝卜的小菜蛾各发育阶段的存活率最低。小菜蛾对各供试寄主实际危害量占理论危害量的百分比为油菜(71.8%)白菜(70.8%)萝卜(53.0%)。表明同一时间内,小菜蛾对油菜危害程度最大,白菜次之,萝卜最小。     

Evidence of <Emphasis Type="Italic">olive mild mosaic virus</Emphasis> transmission by <Emphasis Type="Italic">Olpidium brassicae</Emphasis>

Transmission of three strains of OMMV by an Olpidium sp. was evaluated and compared. The three strains were 1) an OMMV wild type (WT) recovered from olive trees, 2) an OMMV variant (L11) obtained after 15 serial passages of single local lesions induced in Chenopodium murale plants, and 3) a construct OMMV/OMMVL11 in which the coat protein (CP) gene replaced that of the wild type. A single-sporangial culture derived from Chinese cabbage (Brassica pekinensis) used as a bait plant grown in soil of an olive orchard, was identified as Olpidium brassicae based on the size and sequence of the generated amplicon in PCR specific tests. Each of the three virus strains was soil transmitted to cabbage roots in the absence of the fungus at similar rates of 30 to 40%. Separate plant inoculation by O. brassicae zoospores incubated with each viral strain resulted in enhanced transmission of OMMV, reaching 86% of infection whereas that of the other two strains remained practically unaffected at ca. 34%. Binding assays showed that the amount of virus bound to zoospores, estimated spectrophotometrically, was 7% in the case of OMMV, and practically nil in the case of the other two viral strains. Substitution of the coat protein (CP) gene of OMMV by that of the OMMV L11 strain, drastically reduced viral transmissibility in the presence of zoospores to the level of that observed in their absence. Our data shows that OMMV soil transmission is greatly enhanced by O. brassicae zoospores and that the viral CP plays a significant role in this process, most likely by facilitating virus binding and later entrance into the host plant roots.     

新疆北部棉区黄萎病菌种群致病性分化及变异

采用特异性引物PCR检测技术和鉴别寄主法对新疆北部棉区41个棉花黄萎病菌菌系进行检测,以期明确黄萎病菌种群致病性分化及变异。特异性引物(ND1/ND2和D1/D2)PCR检测结果显示,供试菌系中落叶型菌系16个,占39.0%;非落叶型菌系24个,占58.5%;1个菌系未能检测出致病类型。鉴别寄主法测定结果显示:41个供试菌株中致病性强、中、弱的菌系分别为21、12和8个,分别占51.2%、29.3%和19.5%。落叶型菌系的致病力明显高于非落叶型,平均病情指数分别为39.4和25.8。研究表明,无论落叶型菌系和非落叶型菌系,接种后都可产生落叶症状,但落叶的程度有明显差别,其落叶症状的轻重不仅与菌系的致病类型有关,还与品种的抗病性密切相关。     

Root exudates of potato onion are involved in the suppression of clubroot in a Chinese cabbage-potato onion-Chinese cabbage crop rotation

Clubroot, caused by Plasmodiophora brassicae, has emerged as a serious disease threatening cruciferous crop production throughout the world. Crop rotation with non-host species is commonly practised to avoid clubroot, but it is not known whether rotation crops can control clubroot when the resting spores of P. brassicae remain unaffected. Pot experiments were performed to investigate the response of clubroot in Chinese cabbage to crop rotation with potato onion. The results showed that Chinese cabbage rotated with potato onion exhibited less clubroot disease than Chinese cabbage monoculture. Compared with residues from potato onion, the addition of root exudates from potato onion significantly decreased the disease incidence and index of clubroot (p ≤ 0.05). Potato onion root exudates decreased the number of secondary plasmodia of P. brassicae and the expression of the PRO1 gene of P. brassicae. These results suggest that root exudates from potato onion may play an important role in suppressing clubroot in a Chinese cabbage-potato onion-Chinese cabbage rotation system.     

不同基质传代白僵菌对桑天牛幼虫的侵染力与寄主血淋巴酚氧化酶活性的关系

以诱集自土壤中、对桑天牛Apriona germari幼虫具有较高致病性的球孢白僵菌Beauveria bassiana Bb00为出发菌株R0,经反复接种桑天牛幼虫分别获得菌株R1、R2、R3和R4,而通过反复在普通查氏培养基上传代分别获得菌株M1、M2、M3和M4。分别用R0、R2、R4和M2、M4接种桑天牛幼虫,发现在普通培养基上传代会导致菌株致病力降低,而通过桑天牛幼虫传代培养可提高菌株的致病力。桑天牛幼虫感染白僵菌后,其免疫互作使酚氧化酶活性先迅速上升,随后因菌株的适应性增强而开始下降。用菌株M2、M4接种桑天牛幼虫后,其血淋巴酚氧化酶活性比接种R2和R4的高。各菌株引起的桑天牛幼虫酚氧化酶活性出现高峰值的时间与其LT50值具有一定的相关性,反映了各菌株不同的侵染速度;同时各菌株引起的酚氧化酶活性高峰值也与其LC50值高度相关。说明桑天牛幼虫血淋巴中酚氧化酶活性与不同菌株对桑天牛幼虫的毒力具有一定的相关性,同时酚氧化酶活性也可作为反映菌株毒力的一个重要参考指标。     

生防菌Alternaria amaranthi-3对反枝苋的防治效果

反枝苋Amaranthus retroflexus是一种世界性恶性杂草。为了确定微生物除草剂候选菌Alternaria amaranthi-3防除反枝苋的潜力,通过盆栽试验研究了接种浓度、露期和水乳剂型对A.ama-ranthi-3菌株致病力的影响。结果显示,接种浓度显著影响菌株的致病力,在48h露期条件下,接种孢子浓度为105个/mL时,菌株水剂对反枝苋幼苗生长抑制率为35.55%;浓度为107个/mL时,生长抑制率达到75.25%。露期对该菌株的致病力也有较大影响,在不保湿条件下,菌株水剂对反枝苋的生长抑制率为26.43%,而保湿48h处理的生长抑制率达到77.96%。Span80∶Tween80=1∶3的复配乳化剂和大豆油制备的水乳剂可显著降低露期对菌株防效的影响和提高菌株的致病力,无露期时,菌株水乳剂对反枝苋的生长抑制率达到88.35%,显著高于水剂;48h露期条件下,菌株水乳剂处理的生长抑制率为90.59%,而菌株水剂处理为77.96%。表明通过剂型的改进菌株Alternaria amaranthi-3能有效防除反枝苋。     

Pathogenic and genetic diversity in Plasmodiophora brassicae (clubroot) from Japan

Clubroot disease, caused by Plasmodiophora brassicae Woronin, affects various cruciferous crops. Variations in pathogenicity and virulence are present among field populations of P. brassicae. Many races (pathotypes) have been reported in Japan as well as in other countries using various differential systems. Populations can be classified into four pathotypes using two clubroot-resistant (CR) cultivars of Chinese cabbage as differential hosts in Japan. However, it was recently indicated that each population is often heterogenic and composed of multiple genotypes (races or pathotypes). Breakdown in CR cultivars of Chinese cabbage is a problem in some areas of Japan and may contribute to the selective propagation of minor pathogenic genotypes on the CR cultivars. Clubroot has also been recorded on five species of cruciferous weeds in Japan. In particular, clubroot of Cardamine flexuosa is widely distributed in Japan. Some populations of C. flexuosa are often moderately pathogenic on Chinese cabbage and turnip. Therefore, the epidemiological relationship between clubroot of cruciferous crops and that of the weed has been noted but not thoroughly clarified. The relationship between pathogenic and genetic variations has also been examined among populations from cruciferous crops and weeds in Japan. The result implies an interesting genetic relationship among Williams’ races, among pathotypes determined using CR cultivars of Chinese cabbage and among populations from crops and C. flexuosa. This review includes an introduction of the status of studies on pathogenic and genetic diversity in P. brassicae from Japan.     

Colonization by two isolates of Plasmodiophora brassicae with differing pathogenicity on a clubroot-resistant cultivar of Chinese cabbage (Brassica rapa L. subsp. pekinensis)

A commercial clubroot-resistant F₁ cultivar of Chinese cabbage (Brassica rapa L. subsp. pekinensis), Kukai 70, is resistant to an isolate of populations of Plasmodiophora brassicae from Hagi (HG) city and is susceptible to another from Yamaguchi (YMG) city. The degree and frequency of primary and secondary cycle colonization by the isolates in the root hairs and root tissues of cv. Kukai 70 were compared. Seedlings of cv. Kukai 70 were grown in soils amended with inoculum of either HG or YMG and harvested 10 days after inoculation to observe the primary cycle (number of root-hair infections) and 20, 30, and 40 days after inoculation to observe of the secondary cycle (frequency of infected cells and degree of plasmodial development based on the number of nuclei in infected cells). Although more root hairs were infected in HG than in YMG, fewer cells in root tissues including the cortex and medullary rays were infected in HG than in YMG. In addition, YMG developed plasmodia with many nuclei and formed resting spores, whereas plasmodia remained immature with a small number of nuclei in HG and did not form resting spores even by 40 days after inoculation. These results suggest that suppression of plasmodial development during secondary colonization is associated with resistance mechanisms to HG in cv. Kukai 70. Starch did not accumulate (i.e., development of amyloplasts) in HG-infected cells. This may be involved in the suppression of secondary colonization of P. brassicae in the cultivar.     

Relation between pathogenicity and genetic variation within <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

The relation between diversity of pathogenicity on clubroot-resistant (CR) cultivars of Chinese cabbage (Brassica rapa subsp. pekinensis) bred in Japan and DNA polymorphisms in 17 populations of Plasmodiophora brassicae from cruciferous plants was examined by inoculation tests and random amplified polymorphic DNA (RAPD) analysis using 18 arbitrary primers. Four pathotypes (A–D) were identified after inoculation of six CR cultivars of Chinese cabbage in the 17 populations from cruciferous crops. A relatively high level of genetic diversity was also detected among these populations in the RAPD analysis. Although the four pathotypes could not be clearly differentiated using the RAPD data, most populations of three pathotypes had a consistent location on the dendrogram. All pathotype B (virulent on five cultivars except Utage 70) and D (avirulent on all cultivars) populations, which were common in incompatible interactions with cv. Utage 70, were located in a single subcluster. All five pathotype C populations (virulent only on cv. Utage 70) except for one population grouped in another single subcluster. Because four pathotype A populations (virulent on all six cultivars, races 4 and 9) fell in different subclusters, the populations may be genetically polyphyletic. Populations from cruciferous weed Cardamine flexuosa differed remarkably from those from cruciferous crops in pathogenicity on common cultivars of Chinese cabbage and turnip and C. flexuosa, but they grouped in a single cluster with all race 9 populations from crops. Race 9 populations from crops may thus be closely related to populations from the weed rather than to races 1 and 4 from crops.