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     

根肿病生防放线菌B18发酵条件的研究

B18是一株对十字花科根肿病菌Plasmodiophora brassicae具有抑制作用的优良生防放线菌,其活菌以及发酵液都可抑制根肿病菌休眠孢子萌发.为提高其菌体量以及孢子萌发抑制率,本文以菌体生物量和发酵液抑制根肿病菌休眠孢子活性为指标,通过单因素试验和正交设计优化B18的发酵培养基组分以及培养条件.结果 表明:...     

高粱花黑穗病生物学特性及侵染途径研究

 高粱花黑穗病(Ustilago Kenjiana Ito)是我国高粱上发生的一种病害,对产量影响轻微。冬孢子萌发适温是25-30℃,在水滴里萌发率最高,最适pH是6.0-6.5。在不同营养液里以酵母汁及花粉汁里萌发率最高,在人工培养基上病菌生长最适温度是25℃,最适pH是5.9.碳源以葡萄糖、半乳糖、甘露糖和果糖里生长最佳。氮源以蛋白胨和天冬素生长最好.菌瘿黑冬孢子变褐后不经休眠即可萌发。冬孢子在室外可存活一年半,在土壤里经一冬便丧失生活力,室内能存活三年.以冬孢子接种土壤和种子均未发病,属于花器局部侵染,当年侵染当年发病的非系统侵染病害.冬孢子在高粱孕穗或尚未开花的小花护颖与外颖缝隙中侵入子房,而不是在开花期或开花后侵入,也不是从柱头、花柱或其它部位侵入,当年尚能重复侵染.     

土壤温湿度及播种期对玉米丝黑穗病发生的影响

主要研究土壤温度、湿度及播种期与玉米丝黑穗病发生的关系。试验结果表明,土壤温度为21℃,玉米丝黑穗的发病率最高为31.3%,17℃低温下发病率为10.9%~16.7%,温度在24℃时,发病率为3.7%~4.8%;土壤含水量与病害侵染发病有较明显的关系,土壤湿度为15%时利于孢子萌发与侵染,发病率达为31.1%,湿度10%、20%时发病率仅为3.7%~8.8%,干旱和高湿不利于病菌孢子萌发,湿度是决定发病程度的主导因素。早播病害重,晚播病害轻但玉米产量低。     

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.     

榨菜根肿病生防细菌的筛选、鉴定及评价

近年来,由于十字花科根肿病在榨菜上扩展蔓延,严重影响其产量,造成巨大的经济损失。本研究筛选生防菌并研究其对榨菜根肿病的控制作用,为其生物防治应用奠定基础。研究发现茎瘤芥等十字花科蔬菜根肿病菌根经过腐烂处理后,休眠孢子的萌发率可到达70%以上。故本文采用稀释法从榨菜根肿病菌根中分离筛选出一株可刺激休眠孢子萌发的菌株PB19。并测定了PB19与根肿病菌互作的最适温度和最适pH,温室盆栽试验和大田小区试验测定菌株PB19对榨菜根肿病的防治效果。最后将纯化后的菌株送至中国食品发酵工业研究院微生物检测中心(FMIC)进行种属鉴定。结果表明:PB19与根肿菌互作的最适温度为20~25℃,最适pH为5.5~7.0。温室盆栽结果显示PB19对榨菜根肿病的防治效果为71.96%。而大田试验结果显示PB19对榨菜根肿病的防治效果为18.04%。FMIC的鉴定结果显示PB19为假单胞菌属Pseudomonas sp.。     

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?     

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。     

葡萄白腐病(Coniothyrium diplodiella(Speg.)Sacc.)的研究——Ⅱ、葡萄白腐病田间侵染规律的初步探讨

葡萄白腐病菌侵染的主要条件是伤口和充分潮湿。对葡萄的任何绿色组织,通过伤口,都能发生侵染。在高湿度环境下,或在叶片吐水之际,水孔能成为侵染的途径。在葡萄穗上,它们是不可能作为伤口被肉眼分辨出来的,然而穗梗上的自然伤口足够为病菌所入侵。白腐病菌的孢子萌发和入侵,是与潮湿密切相关的。土壤由于下雨或灌溉而充分湿润,可由下列两条途径引起侵染:(1)水膜凝聚引起营养物质外溢,从而促使孢子萌发,以至进一步入侵。(2)由于葡萄的根压增高而引起叶片吐水,给病菌提高水、营养物质的供应量,导致入侵。     

不同条件下韭菜汁对香蕉枯萎病菌4号小种活性抑制的研究

研究了不同温度、pH值、杀虫剂和杀菌剂的影响下,韭菜汁对香蕉枯萎病菌4号小种的抑制作用,以确定韭菜汁在不同条件影响下对病菌的活力。结果表明,在试验的各温度处理后,55℃以上的温度对韭菜汁的成分有破坏作用,菌落的生长受到显著影响。韭菜根汁对病菌孢子的抑制作用不受pH值的影响。酸性至弱碱性情况下,韭菜茎汁对病菌孢子萌发的抑制率为100%;在强碱性条件下,韭菜茎汁中物质的活性促进了孢子的萌发。供试杀虫剂和杀菌剂对韭菜汁的成分无明显破坏作用。     

Suppression of clubroot (Plasmodiophora brassicae) development in Arabidopsis thaliana by the endophytic fungus Acremonium alternatum

This study investigated the ability of an endophytic fungus Acremonium alternatum to reduce clubroot formation in the model plant Arabidopsis thaliana, which is highly susceptible to Plasmodiophora brassicae . Quantitative PCR demonstrated that A. alternatum colonized the P. brassicae -infected roots and shoots of the host plant. When Arabidopsis plants were co-inoculated with P. brassicae and A. alternatum , gall formation was reduced as shown by the reduction of the disease index (DI) by up to 50% compared to plants only infected with P. brassicae, whereas the infection rate was lowered by about 20% only in several, but not all, experiments. Clubroot was similarly suppressed when plants were inoculated with autoclaved A. alternatum spores or spore extracts, showing that viable spores were not needed. However, A. alternatum spores did not inhibit P. brassicae resting spore germination. Compared to the normal root galls, the smaller root galls on A. alternatum -inoculated plants contained fewer resting spores of the clubroot pathogen. It was thus hypothesized that inoculation with A. alternatum delayed the development of P. brassicae . Using quantitative RT-PCR to monitor the expression of P. brassicae genes differentially expressed during the development of the disease, a delayed pathogen development was corroborated. Furthermore, greenhouse experiments identified a time window in which the endophyte had to be administered, where the latest effective time point was 5 days before inoculation with P. brassicae and the optimum treatment was to administer A. alternatum and P. brassicae at the same time. These results indicate that A. alternatum and perhaps similar endophytes could be useful for the management of clubroot disease.     

Effects of cyazofamid against Plasmodiophora brassicae Woronin on Chinese cabbage

Cyazofamid (4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulfonamide) is a novel fungicide with high levels of activity against Oomycetes fungi and Plasmodiophora brassicae Woronin. The effects of cyazofamid were investigated against P. brassicae, the causal agent of clubroot disease in Chinese cabbage. Cyazofamid at 0.3 mg litre(-1) inhibited resting spore germination of this pathogen by about 80%. Cyazofamid at 3-10 mg litre(-1) exhibited fungicidal activity to resting spores of P. brassicae 1-10 days after treatment. When cyazofamid was applied to infested soil, both root-hair infections and club formation caused by P. brassicae were strongly inhibited at 1-3 mg kg(-1) dry soil. These results suggest that cyazofamid directly inhibits resting spore germination, thereby leading to the inhibition of root-hair infection and club formation. Cyazofamid at 3 mg kg(-1) dry soil also exhibited complete control of clubroot disease. The effect of broadcast soil application using a dust formulation at 2 kg AI ha(-1) (equivalent to 1.3 mg AI kg(-1) dry soil), and plug seedling tray application by a suspension concentrate formulation at 200 and 400 mg AI tray(-1) (30 x 60 x 4 cm3) against P. brassicae was also evaluated. Cyazofamid exhibited good efficacy against the pathogen. The sequential treatment including plug seedling tray application with cyazofamid and pre-plant broadcast soil application with the fungicide fluazinam also exhibited excellent levels of control. These results indicate that cyazofamid has a high potential to be an effective fungicide for the control of clubroot disease.     

Suppression of clubroot disease under neutral pH caused by inhibition of spore germination of Plasmodiophora brassicae in the rhizosphere

To elucidate the mechanism of clubroot suppression under neutral soil pH, a highly reproducible germination assay system under soil culture conditions was designed based on the hypothesis that germinated spores of Plasmodiophora brassicae could be identified by the absence of a nucleus (i.e. having released a zoospore to infect a root hair of the host plant). Brassica rapa var. perviridis seedlings were inoculated with a spore suspension of P. brassicae at a rate of 2·0 × 10⁶ spores g⁻¹ soil and grown in a growth chamber for 7 days. The spores were recovered from rhizosphere and non-rhizosphere soils and stained with both Fluorescent Brightener 28 (cell-wall-specific) and SYTO 82 orange fluorescent nucleic-acid stain (nucleus-specific stain). Total numbers of spores were counted under UV-excitation, and spores with a nucleus that fluoresced orange under G-excitation were counted. The significant increase in the percentage of spores without a nucleus (germinated spores) in the rhizosphere after 7 days' cultivation and the correlation with root-hair infections validated the assay system. Applications of calcium-rich compost or calcium carbonate to neutralize the soil significantly reduced the percentage of germinated spores in the rhizosphere, as well as the number of root-hair infections. The present study provides direct evidence that the inhibition of spore germination is the primary cause of disease suppression under neutral soil pH.     

Usefulness of nonhost plants in managing Plasmodiophora brassicae

Germination of resting spores of Plasmodiophora brassicae , causal agent of clubroot in crucifers, may be stimulated by certain nonhost plants. Without a host plant to infect, such germination would lead to a reduced persistence of resting spores in the soil. The effect of four nonhost plants on P. brassicae was investigated in a 3-year field experiment and a 14-month glasshouse experiment. Three of the plant species used, leek ( Allium porrum ), winter rye ( Secale cereale ) and perennial ryegrass ( Lolium perenne ), have been reported to stimulate resting spore germination, while the fourth, red clover ( Trifolium pratense ), does not. In the field experiment, none of the plant species reduced the concentration of P. brassicae in soils when tested with bioassay plants (Chinese cabbage, Brassica rapa var. pekinensis ). In the glasshouse experiment, there was a lower disease level in all plant treatments compared with the plant-free control following incorporation and decomposition of plant roots. At this time, pH in the soils with plant treatments was higher than that in the control soil. There were no indications of a species-specific interaction between any of the nonhost plants investigated and P. brassicae , and it cannot be concluded that any of them would be useful in the sanitation of P. brassicae -infested soils within short time periods.     

甘蓝根肿病的人工接种体系与抗源材料筛选

试验分别比较了不同接种方法、不同接种浓度以及不同土壤pH条件下人工接种甘蓝根肿病后甘蓝的发病情况,筛选出一套甘蓝根肿病苗期抗性鉴定与评价的最佳接种体系:最佳接种方法为菌土法;最佳孢子接种浓度为1.6×108个/g基质,最佳土壤pH为6.0。利用最佳人工接种体系,对东北农业大学甘蓝课题组提供的20份甘蓝材料进行抗性鉴定,其中抗病(R)材料3份,中抗(MR)材料9份,感病(S)材料7份,高度感病(HS)材料1份。     

模拟酸雨对小麦条锈病流行学组分的影响

为明确酸雨对小麦条锈病菌孢子萌发及病害流行学组分的影响,采用不同酸度的模拟酸雨(p H值分别为2.5、3.5、4.5和5.6)处理小麦条锈病菌Puccinia striiformis f.sp.tritici生理小种CYR32和CYR33的夏孢子,并将不同酸度模拟酸雨处理后的夏孢子接种小麦感病品种铭贤169,采用组分分析法研究酸雨对病害发展过程的影响。结果表明,随着模拟酸雨酸度的增强,生理小种CYR32和CYR33的夏孢子萌发率均显著降低,萌发24 h后对照组生理小种CYR32和CYR33的夏孢子萌发率分别为93.7%和79.8%,在p H 3.5模拟酸雨处理后萌发率仅为13.1%和8.6%。温室内接种试验结果显示,不同酸度模拟酸雨对生理小种CYR32和CYR33的侵染概率、潜育期和病情指数均有显著影响,对生理小种CYR32和CYR33病斑扩展率影响不显著;与对照相比,p H 3.5模拟酸雨处理后生理小种CYR32和CYR33的侵染概率分别下降79.9%和79.8%。结果表明强酸雨(p H 3.5)显著延长小麦条锈菌潜育期,减少病斑产孢量,降低病情指数和病害进展曲线下面积。     

Nitrogen supply exerts a major/minor switch between two QTLs controlling Plasmodiophora brassicae spore content in rapeseed

Clubroot, caused by Plasmodiophora brassicae, is a worldwide disease affecting Brassica. Until now, the detection of genetic factors (QTLs) implicated in clubroot resistance has been based on estimates of disease index. However, as the amount of resting spores released in soil after club disintegration influences clubroot epidemics and resistance‐breaking dynamics, its genetic control may deserve specific attention. In a previous report, it was shown that nitrogen fertilization modulated quantitative partial resistance toward clubroot symptom development in rapeseed. The present work aimed to identify genetic factors involved in the control of resting spore production and to assess their regulation by nitrogen supply. A flow cytometer method was adapted for rapidly estimating resting spore content in a large series of samples. Linkage analysis was conducted to detect QTLs implicated in resting spore production in a Brassica napus doubled haploid progeny from the cross Darmor‐bzh × Yudal. DH lines inoculated with the P. brassicae isolate eH were grown under low‐ and high‐nitrogen supply. Under low‐nitrogen conditions, resting spore production was reduced compared to high‐nitrogen conditions, regardless of genotypes. Genetic architecture controlling resting spore production and clubroot symptom development was similar. Under high‐nitrogen conditions, resting spore production was controlled by one major QTL (C09a) and a few small‐effect QTLs. By contrast, two major QTLs (C02 and C09a) controlled resting spore production under low‐nitrogen conditions. This work highlighted a large see‐saw effect between the relative contribution of the C09a QTL (high effect under high‐nitrogen conditions) and the C02 QTL (high effect under low‐nitrogen conditions), with possible implications in resistance breeding.     

红海榄赤斑病病原菌鉴定及其生物学特性研究

 本文对来自红海榄(Rhizophora stylosa)赤斑病同一病斑上获得的2个菌株PⅠ-1和PⅡ-3进行了鉴定和生物学特性研究。结果表明,PⅡ-3菌株的致病力比PⅠ-1的稍强;2个菌株均具有一定的寄主专化性。经形态特征观察与rDNA-ITS序列分析,2个菌株均鉴定为异色拟盘多毛孢(Pestalotiopsis versicolor)。生物学特性测定结果表明,温度、pH值和光照对2个菌株菌丝生长及孢子萌发的影响差异不大,生长温度均为10~32℃(最适25℃),pH值为3.0~10.9(最适4.0);孢子萌发温度均为10~35℃(最适25~28℃),pH值为2.5~8.0(最适3.0);黑暗条件有利于2个菌株菌丝生长和孢子萌发。但2个菌株的产孢特性存在明显差异,其中PⅠ-1产孢的最适温度为28℃而PⅡ-3为20℃;PⅠ-1产孢的最适pH值为5.1而PⅡ-3为9.0;PⅠ-1产孢对光照不敏感,而PⅡ-3在黑暗条件下几乎不产孢。另外,在相似条件下,PⅠ-1产孢量比PⅡ-3大,而PⅡ-3菌丝生长速度比PⅠ-1快,孢子萌发率也比PⅠ-1高。由此可见,2个菌株PⅠ-1和PⅡ-3虽属于同一个种,但某些生物学特性存在一定的差异。     

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.     

APN1的原核表达及其与Cry1Ac蛋白体外结合

本研究分析了Cry1Ac蛋白与棉铃虫BBMVs的结合情况;并将棉铃虫apn1基因的全长和片段在大肠杆菌Rosetta(DE3)中进行表达,利用Ligand blotting技术分析原核表达的APN1全长及4个片段与Cry1Ac蛋白的结合情况。结果表明:Cry1Ac蛋白可以与棉铃虫的BBMVs结合;在大肠杆菌中表达的棉铃虫受体蛋白APN1的全长、片段H1(33~257)、H2(258~547)和H3(548~798)可以与Cry1Ac蛋白结合,其中片段H3是首次发现可以与Cry1Ac蛋白结合。本研究的结果也为其他Bt蛋白受体的体外功能研究提供新的借鉴。