利用荧光定量PCR检测禾谷丝核菌的相对生物量

小麦纹枯病是以禾谷丝核菌(Rhizoctonia cerealis)侵染为主的小麦土传病害。为建立检测禾谷丝核菌在寄主小麦(Triticum aestivum)中的相对生物量的可靠方法,促进小麦抗纹枯病机制的研究,本研究克隆了禾谷丝核菌肌动蛋白基因RcActin的部分(3′端)cDNA,并设计了RcActin的特异引物。该引物不仅能区分禾谷丝核菌与寄主小麦,还能区分全蚀病菌(Gaeumannomyces graminis)、根腐病菌(Bipolaris sorokiniana)和立枯丝核菌(Rhizoctonia solani)等常见小麦土传病害的病原菌,表明该引物能用于小麦纹枯病的分子检测,也能用于相对表达量的测定。利用相对定量法,以RcActin相对于寄主管家基因的相对表达量作为禾谷丝核菌相对生物量的指标,结果表明,此方法能准确反映禾谷丝核菌在寄主中的相对生物量和对小麦纹枯病抗性程度进行快速鉴定。     

禾谷丝核菌环介导等温扩增检测技术体系的建立

为建立简便、快速和灵敏的小麦纹枯病菌——禾谷丝核菌Rhizoctonia cerealis的分子检测技术体系,基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以核糖体DNA内转录间隔区(internal transcribed spacer,ITS)为靶标序列,设计并筛选特异性引物,建立禾谷丝核菌的LAMP检测方法,并对其特异性、灵敏度和应用效果进行测定。结果表明,基于筛选获得的4条禾谷丝核菌特异性引物建立的LAMP方法能够从15种植物病原菌中特异性地检出禾谷丝核菌,近缘种立枯丝核菌Rhizoctonia solani和引致早期症状易混淆病害的病原菌禾顶囊壳菌Gaeumannomyces graminis、麦根腐平脐蠕孢菌Bipolaris sorokiniana和禾谷镰孢菌Fusarium graminearum均未检出;该LAMP方法在日光下的灵敏度为10^-1 ng/μL,在蓝光下的灵敏度为10^-3 ng/μL;对发病组织的检测显示该LAMP方法能从人工接种的小麦发病组织中...     

小麦苗期抗纹枯病鉴定方法的改良及抗病品种筛选

<正>小麦纹枯病又称尖眼斑(点)病,是由禾谷丝核菌(Rhizoctonia cerealis)和立枯丝核菌(Rhizoctonia solani)引起的一种以土壤传播为主的小麦真菌病害~([1])。引起小麦纹枯病的病原菌主要是禾谷丝核菌的第一菌丝融合群(CAG-1)~([2～4])。在自然条件下,病原菌首先侵染寄主植株基部叶鞘,随着     

青岛市小麦纹枯病病原菌鉴定及其病害分析

采用组织分离法对青岛地区小麦纹枯病病原菌进行了分离,结合菌物形态学特征、rDNA-ITS和特异性引物的PCR扩增等分子检测对病原菌进行鉴定。结果表明,引起青岛地区小麦纹枯病的病原菌主要为禾谷丝核菌(Rhizoctonia cerealis)CAG-1(AG-D)融合群。据田间调查结果 ,分析了病害暴发的原因,提出了以改善农田生态条件为基础,结合药剂防治以及利用抗(耐)病品种的生产策略。     

河南省小麦纹枯病菌的群体组成及其致病力分化研究摇

纹枯病已成为我国小麦生产的主要障碍之一.许多学者研究表明,我国小麦纹枯病菌优势菌群为禾谷丝核菌(Rhizoctonia cerealis),其中主要为AG-D融合群,其次还包括AG-A、AG-R、AG-E、AG-Bo融合群和立枯丝核菌(R.solani)的AG-2、AG-4及AG-5融合群等[1].     

禾谷丝核菌(Rhizoctonia cerealis)原生质体制备与再生的研究

禾谷丝核菌(Rhizoctonia cerealis)是引起我国小麦纹枯病的主要致病菌。为了建立高效稳定的禾谷丝核菌遗传转化体系,本试验比较研究了不同细胞壁降解酶、酶液浓度、酶处理温度和时间等因素对禾谷丝核菌原生质体制备的影响,利用正交设计试验优化了原生质体再生条件。结果表明,液体培养6d的菌丝,采用15mg/mL溶壁酶+10mg/mL蜗牛酶组成的混合酶液,30℃下酶解4h,可以获得较高的原生质体释放量,可达到3.0×106个/mL;禾谷丝核菌原生质体再生的最佳条件是以SuTC缓冲液作为渗透压稳定剂悬浮原生质体,采用单层混菌法接种于TB3再生培养基,原生质体再生率可达到58.6%。禾谷丝核菌原生质体制备和原生质体再生条件的优化,为深入研究禾谷丝核菌生长发育的分子遗传学基础和进一步探索小麦纹枯病的致病机理奠定了基础。     

禾谷丝核菌基因组SSR标记的开发及评价

群体遗传学研究对于了解病原菌的流行、变异及进化规律具有重要意义。但是到目前为止,对小麦纹枯病菌禾谷丝核菌群体遗传结构的了解并不深入,缺乏有效的SSR标记是主要原因。本研究根据禾谷丝核菌全基因组序列进行了微卫星位点搜索并设计SSR引物,通过电泳筛选和测序验证,最终筛选出12个多态性较好且稳定可靠的SSR标记。利用这些标记对收集自我国安徽、江苏、河南三省的23个禾谷丝核菌菌株进行了多态性分析,结果发现禾谷丝核菌基因组中长片段微卫星位点较少。12对引物扩增出的等位基因数平均为6.1个,期望杂合度平均为0.651,观测杂合度平均为0.508,表明这些标记具有较高的多态性,能够满足禾谷丝核菌群体遗传学研究需要。本研究为进一步进行禾谷丝核菌的多样性、群体遗传结构分析及进化学研究提供了有效工具。     

江苏省大、小麦纹枯病病原学的初步研究

 1984至1986年间,采集江苏各地大、小麦纹枯病标样,分离获得丝核菌属菌株分别为23个和50个。经比较鉴定其培养特性,菌丝形态、隔膜孔器以及细胞核数目,结合菌丝融合测试,50个小麦纹枯丝核菌菌株中除2个属于立枯丝核菌(Rhizoctonia solani Kühn)AG₅群外,其余均为禾谷丝核菌(R.cetea-lis Vander Hoeven),其中CAG₁、CAG₃、CAG₆、AGC₁分别为44、1、1和2个菌株。在23个大麦纹枯丝核菌菌株中,除2个属于立枯丝核菌AG₅群外,其余均为禾谷丝核菌,其中CAG₁、CAG₂、AGE的菌株分别为18、1、1、另有一菌株不属所测融合群。
人工交互接种试验表明:来源于大麦和小麦的禾谷丝核菌CAG₁群菌株对于大、小麦均有极强的致病力;立枯丝核菌AG₅群菌株次之,也有一定致病力;其他融合群的菌株致病力较弱。因此,引致江苏省大小麦纹枯病的主要病原菌为禾谷丝核菌(R.cerealis)的CAG₁群。     

我国小麦纹枯病的研究动向

我国小麦产区,近年来小麦纹枯病在广大小麦产区,特别是主产和高产的黄淮麦区,迅速扩散蔓延,危害严重,已上升为影响小麦高产、稳产的主要病害。一般病因减产１０％左右,重者可达３０％～５０％,甚至更高,给小麦产量带来了巨大损失。 小麦纹枯病菌（Ｒｈｉｚｏｃｔｏｎｉａ ｃｅｒｅａｌｉｓ）属丝核菌属,是一种重要的土传性禾谷类病害,种名为禾谷丝核菌。 我国小麦纹枯病的病原菌主要是禾谷丝核菌的ＣＡＧ－１融合群,其次是立枯丝核菌（Ｒ．ｓｏｌａｎｉ）的ＡＧ－５和ＡＧ－４融合群。 我国早在１９７５年就有小麦纹枯病的报道,…     

10种杀菌剂及其复配剂对小麦纹枯病菌的室内毒力

小麦纹枯病是由禾谷丝核菌(Rhizoctonia cerealis)引起的冬小麦种植区的一种重要土传真菌病害.为筛选防治小麦纹枯病的高效低毒杀菌剂,缓解和治理生产中病原菌对药剂的抗性,在室内离体条件下采用菌丝生长速率法测定了10种杀菌剂(原药)对禾谷丝核菌的毒力.结果表明:98%戊唑醇原粉、95%氟环唑原粉、96%烯唑醇原粉、96%三唑醇原粉、97%吡唑醚菌酯原粉、95%丙环唑原油的EC50分别为0.115,0.158,0.237,0.432,0.652和1.426mg/L,表明小麦纹枯病菌对上述杀菌剂比较敏感;95%苯菌灵原粉、97%咪鲜胺原油和98%噻菌灵原粉的EC50分别为8.034,9.225和14.894mg/L,病菌对药剂的敏感程度相对较低;97.56%甲基硫菌灵原粉的EC50为587.362mg/L,抑菌活性较差.将不同作用机理的杀菌剂吡唑醚菌酯分别与烯唑醇、三唑醇、噻菌灵和甲基硫菌灵按照有效成分1∶1的比例复配,联合毒力测定和评价结果表明,上述复配药剂对抑制小麦纹枯病菌具有明显增效作用.     

Damping-off of sugar beet caused by Rhizoctonia cerealis

Rhizoctonia cerealis was isolated from diseased sugar beet seedlings collected from crops in Ireland. In pathogenicity tests, isolates of R. cerealis from beet seedlings, and from sharp eyespot lesions on wheat crops, caused severe damping-off of sugar beet. Isolates from beet seedlings also caused symptoms of sharp eyespot on wheat.     

中国小麦纹枯病化学防治研究进展

随着秸秆还田等耕作栽培措施的推广,中国小麦纹枯病发生日趋严重,对小麦的高产、稳产造成了很大威胁。由于缺乏免疫及高抗病性小麦品种,生产中对纹枯病一直采用播期拌种及春季喷雾相结合的化学防治方法。文章总结了当前中国小麦纹枯病的发生现状及主要病原;评述了三唑类药剂对纹枯病菌的毒力及对纹枯病的防治效果,介绍了生产中小麦纹枯病菌对三唑类药剂的抗药性现状及机理,分析了三唑类药剂对小麦的安全性;同时阐述了井冈霉素、甲基立枯磷及其他种类药剂在小麦纹枯病综合防治中的应用;指出小麦纹枯病化学防治的发展方向应是将生防菌剂同化学药剂相结合,实现生物防治与化学防治的协同应用。     

Development and use of a PCR assay to detect Rhizoctonia cerealis, the cause of sharp eyespot in wheat

Random amplified polymorphic DNA assays were carried out on a range of isolates of Rhizoctonia cerealis to identify markers common to all isolates. Two fragments were isolated, cloned and used to probe Southern blots of DNA from R. cerealis and isolates from a range of anastomosis groups of Rhizoctonia solani. The two fragments hybridized specifically to DNA of R. cerealis and not to DNA of any of the isolates of R. solani. Both fragments were partially sequenced and two pairs of primers were generated for use in the polymerase chain reaction (PCR). Amplification of a fragment of the anticipated size occurred following PCR of all isolates of R. cerealis and not from any of a range of fungal species associated with disease of the stem base of cereals. The primer pairs for R. cerealis were also used, along with those for Microdochium nivale and W and R-type of Pseudocercosporell herpotrichoides , to deled these pathogens in extracts from field-grown wheat plants exhibiting symptoms of sharp eyespot, eyespot, foot rot or a combination of the diseases. No relationship was found between visual disease assessment of sharp eyespot at growth stage 37 and the results of PCR However, the results of PCR and visual disease assessment at growth stage 75 were similar, indicating that visual disease assessment may not be reliable until later growth stages. This system offers the potential to detect the presence of R. cerealis in cereals and avoid problems commonly associated with conventional diagnosis of this disease and isolation of the pathogen.     

河南省小麦纹枯病菌对三唑酮的敏感性

正小麦纹枯病是一种世界分布的土传真菌病害。近年来该病在我国冬小麦种植区发生普遍,已成为小麦高产、稳产的重大障碍~([1])。河南省是我国小麦主产区之一,近几年小麦纹枯病的发生面积连续在300万hm~2左右,危害严重~([2])。目前,尚未发现高抗纹枯病的小麦品种,控制该病害主要依靠药剂种子处理结合春季喷雾来进行,所用药剂包括井冈霉素和以三唑酮为代表的三唑类化学药剂。随着     

The escalating threat of Rhizoctonia cerealis, the causal agent of sharp eyespot in wheat

Rhizoctonia cerealis, the causal agent of sharp eyespot on wheat, was not considered to be an important pathogen for many years. Recently, the disease has become endemic in many countries except for South America. The disease has created a new threat to world wheat production because the damage of wheat sharp eyespot has become increasingly severe. In this paper, previous studies on this pathogen, including the disease geographical distribution, pathogen identification, life cycle, symptoms, favourable environmental conditions, effects on wheat yield and control strategy, are reviewed. Such information will be helpful in management of sharp eyespot.     

Infection of potato and wheat by isolates of Rhizoctonia solani and R. cerealis

Isolates of Rhizoctonia were obtained from potato crops with stem canker or black scurf and from cereal crops with sharp eyespot. Those with many nuclei per cell, wide cells, darkening colonies and fast growth were assigned to R. solani ; those with two nuclei per cell, narrow cells, pale colonies and slow growth were assigned to R. cerealis . Only R. solani was obtained from potatoes and only R. cerealis from cereals. On young plants in the glasshouse, the isolates of R. solani infected potato substantially but not wheat; R. cerealis infected wheat substantially and potato slightly. This host preference was shown at temperatures between 10 and 25°C in growth rooms. R. solani on potato caused more disease with increasing temperature; no trend with temperature was observed for R. cerealis on wheat.     

Visual disease and PCR assessment of stem base diseases in winter wheat

Stem base disease (eyespot, sharp eyespot and brown foot rot) was assessed visually and by the polymerase chain reaction (PCR) technique on single plants sampled at four-week intervals in two crops of winter wheat grown in the UK in 1992–3. PCR assays were conducted for Fusarium avenaceum , F. culmorum , both varieties of Microdochium nivale , both eyespot-causing species of Tapesia and Rhizoctonia cerealis . PCR diagnoses were compared with visual diagnoses at each time point. Eyespot was caused principally by T. acuformis (R-type) and developed rapidly late in the season. Visual diagnoses of eyespot were largely confirmed by PCR but T. acuformis was detected in many plants lacking eyespot symptoms. R. cerealis was detected at relatively low incidences on both sites, and sharp eyespot visual diagnoses did not correlate with the incidence of any of the pathogens assayed by PCR. Brown foot rot, caused principally by Microdochium nivale var . majus , accumulated earlier in the season than eyespot. Overall, visual diagnoses of stem base disease coincided poorly with PCR data until after growth stage (GS) 30.     

小麦纹枯病流行预测的研究

作者根据小麦纹枯病在冬麦区发生具有明显阶段性的特点,利用1984～1993年连续10年在纹枯病常发区山东省文登市的多品种、多地点的病情系统调查数据,采取统计学和流行学相结合的方法,组建了具有预测秋苗冬前发病率和返青后生长期病情指数的回归模型。并通过对小麦纹枯病普遍率和病情指数相关性的研究,将二阶段的模型耦联,完成对小麦整个生长期的病害流行动态全过程的预测。     

小麦纹枯病菌中的dsRNA及其与菌株生物学特性的相关性

对采集自我国山东、河南、江苏、安徽4省的21株小麦纹枯病菌菌丝中的dsRNA进行了检测,研究了dsRNA条带与菌株菌落形态、生长速度、对小麦的致病力等生物学特性之间的相关性。结果表明,从16个菌株中检测到dsRNA条带,dsRNA的大小和数量存在丰富的多样性。致病力测定结果表明这21个小麦纹枯病菌菌株的致病力差异显著。目前暂未发现小麦纹枯病菌中dsRNA的类型和数量与菌株的菌落形态、生长速度及致病力等性状存在明显的相关性,但是致病力较弱的菌株中dsRNA条带的多样性更丰富。本研究为今后发掘小麦纹枯病菌中的低毒真菌病毒作了初步的探索。