Genome sequencing reveals the evolution and pathogenic mechanisms of the wheat sharp eyespot pathogen Rhizoctonia cerealis

The necrotrophic fungus Rhizoctonia cerealis is the causal agent of devastating diseases of cereal crops including wheat(Triticum aestivum).We present a high-quality genome assembly of R.cerealis Rc207,a virulent strain causing wheat sharp eyespot.The assembly(56.36 Mb)is composed of 17.87%repeat sequences and 14,433 predicted protein-encoding genes.The Rc207 genome encodes a large and diverse set of genes involved in pathogenicity,especially rich in those encoding secreted proteins,carbohydrateactive enzymes(CAZymes),peptidases,nucleases,cytochrome P450,and secondary metabolismassociated enzymes.Most secretory protein-encoding genes,including CAZymes,peroxygenases,dehydrogenases,and cytochrome P450,were up-regulated during fungal infection of wheat.We identified 831 candidate secretory effectors and validated the functions of 10 up-regulated candidate effector proteins.Of them,nine were confirmed as necrotrophic pathogen’s effectors promoting fungal infection.Abundant potential mobile or plastic genomic regions rich in repeat sequences suggest their roles in fungal adaption and virulence-associated genomic evolution.This study provides valuable resources for further comparative and functional genomics on important fungal pathogens,and provides essential tools for development of effective disease control strategies.

Changes in Activities of Antioxidant-Related Enzymes in Leaves of Resistant and Susceptible Wheat Inoculated with Rhizoctonia cerealis

In this study, the changes in activities of peroxidase （POD）, glutathione S-transferase （GST）, catalase （CAT）, and superoxide distmuase （SOD） in leaves of four wheat cultivars, including resistant cultivars Shannong 0431 and Shanhongmai, a mildsusceptible cultivar Lumai 21, and a susceptible cultivar Wenmai 6, were investigated to explore if these antioxidantrelated enzymes were involved in wheat defense response to Rhizoctonia cerealis attack. Results showed that the activities of these enzymes in different wheat cultivars varied greatly after challenging with R. cerealis. The POD activities in resistant cultivars Shannong 0431 and Shanhongmai increased much quicker after the inoculation and displayed much higher activity than that in susceptible cultivar Wenmai 6. In contrast, POD activity showed a slow accumulation and a delayed peak time. The activities of GST and CAT in Shannong 0431 and Lumai 21 increased obviously within 48 h post inoculation （hpi）, whereas SOD activity decreased dramatically within 24 hpi in Shannong 0431 and 24-48 hpi in Lumai 21. In susceptible cultivar Wenmai 6, the SOD and CAT activities didn’t change obviously, whereas the activity of GST was decreased. The activities of CAT and GST in Shanhongmai did not show significant difference compared with those in Wenmai 6. Results suggested that POD activity may be positively associated with wheat defense to R. cerealis.

The wheat receptor-like cytoplasmic kinase TaRLCK1B is required for host immune response to the necrotrophic pathogen Rhizoctonia cerealis

Receptor-like cytoplasmic kinases(RLCKs)represent a large family of proteins in plants.In Arabidopsis and rice,several RLCKs in subfamily VII(RLCKs-VII)have been implicated in pathogen-associated molecular pattern-triggered immunity and basal resistance against bacterial and fungal pathogens.However,little is known about roles of RLCKs-VII of the important crop common wheat(Triticum aestivum)in immune responses.Here,we isolated a RLCK-VII-encoding gene from wheat,designated as TaRLCK1B,and investigated its role in host immune response to infection of a necrotrophic fungus Rhizoctonia cerealis that is a major pathogen of sharp eyespot,a destructive disease of wheat.RNA-sequencing and RT-qPCR analyses showed that transcriptional level of TaRLCK1B was significantly higher in sharp eyespot-resistant wheat cultivars than in susceptible wheat cultivars.The gene transcription was rapidly and markedly elevated in the resistant wheat cultivars by R.cerealis infection.The TaRLCK1B protein was closely related to OsRLCK176,a rice resistance-related RLCKs-VII,with 84.03%identity.Virus-induced gene silencing plus wheat response to R.cerealis assay results indicated that silencing of TaRLCK1 impaired resistance to R.cerealis.Meantime,silencing of TaRLCK1 significantly elevated both the content of H2 O2(a major kind of reactive oxygen species,ROS)and the transcriptional level of the ROS-generating enzyme-encoding gene RBOH,but repressed the expression of the ROS-scavenging enzyme-encoding gene CAT1 at 18 hours after inoculation(hai)with R.cerealis.Taken together,these data suggested that TaRLCK1B was required for the early immune response of wheat to R.cerealis through modulating ROS signaling in wheat.

The M43 domain-containing metalloprotease RcMEP1 in Rhizoctonia cerealis is a pathogenicity factor during the fungus infection to wheat

Wheat(Triticum aestivum L.)is an important staple crop for global human.The necrotrophic fungus Rhizoctonia cerealis is the causal pathogen of sharp eyespot,a devastating disease of wheat.Herein,we identified RcMEP1,a zinc metalloproteaseencoding gene from R.cerealis genomic sequences,and characterized its pathogenesis function.RcMEP1 expressed at markedly-high levels during R.cerealis infection process to wheat.The predicted protein RcMEP1 comprises of 287 amino acid residues and contains a signal peptide and a M43 metalloprotease domain harboring the active site motif(HEVGHWLGLYH).The assays of Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana leaves indicated that RcMEP1 is an apoplastic elicitor of cell death,and that the predicted signal peptide functions and is required for secretion and cell death-induction.The purified RcMEP1 protein and its M43 domain peptide were individually able to induce plant cell death and H2 O2 accumulation,and to inhibit expression of host chitinases when infiltrated into wheat and N.benthamiana leaves,while the M43 domain-deleting peptide and negative control lacked the capacity.Moreover,compared with the control pretreatment,the purified RcMEP1 protein or its M43-domain peptide resulted in enhanced pathogenesis in the inoculated wheat,whereas the M43 domain-deleting peptide failed.These results suggest that RcMEP1 acted as an important pathogenicity factor during R.cerealis infection to wheat and that its signal peptide and M43 domain are required for the secretion and pathogenesis of RcMEP1.This study provides insights into pathogenesis role of M43 domain-containing metalloproteases during R.cerealis infection to wheat.

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。

江苏小麦纹枯病菌(Rhizoctonia Cerealis Vander Hoeven)CAG_1群菌株的特性和致病力分化

1989—1990年从江苏省11个市的24个县采集小麦纹枯病株,分离到属于禾谷丝核菌(Rhizoctonia cerealis vander Hoeven)的CAG_1群菌株39个,研究了各菌株在20℃、30℃下的菌丝生长速率,对Cu^(++)(0.5克/升)和Li^+(2克/升)耐性,成株期和幼苗期的致病力以及对三唑酮的敏感性(EC_(50))。结果表明:江苏省禾谷丝核菌CAG_1群内的菌株间存在明显的变异性,特别是对麦苗的致病力差异极为明显。采用模糊逐步聚类法,39个菌株可分成Ⅰ、Ⅱ、Ⅲ类,各类菌株诱发幼苗发病率的平均值分别为1.2％、45.7％和74.8％,显示同一融合群(CAG_1)菌株间存在致病力分化现象。

Introgression of sharp eyespot resistance from Dasypyrum villosum chromosome 2VL into bread wheat

Wheat sharp eyespot, a stem disease caused by the soilborne fungus Rhizoctonia cerealis van der Hoeven,has become a threat to wheat production worldwide. Exploiting resistance resources from wild relatives of wheat is a promising strategy for controlling this disease. In this study, a new wheat–Dasypyrum villosum T2DS·2V#4L translocation line in the background of Chinese Spring(CS) showed stable resistance to R. cerealis. Introgression of the T2DS·2V#4L chromosome into wheat cultivar Aikang 58 by backcrossing produced a marked increase in sharp eyespot resistance in NIL-T2DS·2V#4L in comparison with NILT2DS·2DL, and no detrimental effects of 2V#4L on agronomic traits were observed in the BC2F2, BC2F2:3,and BC2F2:4generations. Flow-sorted sequencing of 2V#4L yielded 384.3 Mb of assembled sequence, and8836 genes were predicted of which 6154 had orthologs in at least one of the 2AL, 2BL, and 2DL arms of CS, whereas 1549 genes were unique to 2V#4L. About 100,000 SNPs were detected in genes of 2V#4L and2DL in 10 sequenced bread wheat cultivars. A Kompetitive Allele Specific Polymerase chain reaction and30 conserved ortholog sequence markers were developed to trace the 2V#4L chromatin in wheat backgrounds. T2DS·2V#4L compensating translocation lines represent novel germplasm with sharp eyespot resistance and the markers will allow rapid detection in breeding programs.

Susceptibility of Wheat Varieties to Soil-Borne <i>Rhizoctonia</i>Infection

Response of 19 wheat varieties cultivated in Hungary varied within large limits to soil borne Rhizoctonia infection. The most frequent symptom, usually leading to damping off was the root neck necrosis. Four significant factors influencing the susceptibility of wheat comprised 71% of total variation but none of them was dominant. The inhibition of development of survivors in Rhizoctonia infested soil correlated with overall susceptibility of variety concerned. The varieties Emese, Kikelet and Palotás are proved to be less susceptible, but none of the varieties could be certified as tolerant. No relationships were revealed between pathogenicity of 26 Rhizoctonia strains studied and their taxonomic position or origin. The anamorph strains of Athelia, Ceratobasidium, Ceratorhiza and Waitea similar to Thanatephorus anamorphs selectively infected the wheat varieties, but the syndromatic pictures were undistinguishable with unarmed eye. R. solani was proved to be more aggressive against germinating wheat than R. cerealis. Nine significant factors influencing the virulence of Rhizoctonia strains comprised 82% of total variation, and six of them influenced exclusively Thanatephorus anamorphs.

Composition of Wheat Rhizosphere Antagonistic Bacteria and Wheat Sharp Eyespot as Affected by Rice Straw Mulching

To assess the effect of rice straw mulching on changes of antagonistic bacteria and the incidence of wheat sharp eyespot, a multi-year field study was performed to compare unmulched plots and the plots mulched with rice straw for two or three years. Bacterial and fungal populations were evaluated in the cultures prepared from the wheat rhizosphere and bulk soils. Rice straw mulching increased the number of pseudomonas colony forming units in wheat rhizosphere and bulk soils. The proportion of total bacteria that were fluorescent pseudomonads was higher in mulched than in unmulched soil. Bacterial isolates antagonistic to Rhizoctonia cerealis were identified using an inhibition zone test. A series of these isolates were typed by partial sequencing of the 16S rRNA gene. Pseudomonads had higher antagonistic activity against R. cerealis than other species, and more than 80% of rhizosphere fluorescent pseudomonads were antagonistic to R. cerealis. The disease indices were lower in the mulched plots than in the unmulched control. These results suggest that rice straw mulching in a rice-wheat rotation increases the number of fluorescent pseudomonads. Additionally, these fluorescent pseudomonads may contribute to the control of wheat sharp eyespot.

实时定量PCR法预测禾谷丝核菌Rhizoctonia cerealis基因组大小

【目的】准确测定基因组大小是进行禾谷丝核菌Rhizoctonia cerealis全基因组序列测定和拼接的基础,本研究旨在利用实时定量PCR方法预测禾谷丝核菌的基因组大小。【方法】首先克隆了禾谷丝核菌R0301菌株翻译延伸因子A基因(tef A)的部分序列,Southern杂交明确该基因在该病菌基因组中为单拷贝。以已测序立枯丝核菌(Rhizoctonia solani)AG1-IA融合群菌株GD118为对照,采用实时定量PCR的方法进行了禾谷丝核菌基因组大小的预测。【结果】实时定量PCR的方法可以比较准确的测定立枯丝核菌基因组的大小,研究首次预测了禾谷丝核菌的基因组大小位于32.2–36.6 Mb之间。【结论】实时定量PCR法是一种快速和简便的预测丝核菌基因组大小的方法。

防治小麦纹枯病菌和玉米茎基腐病菌的药剂筛选及复配增效评价

采用生长速率法,测定不同杀菌剂对小麦纹枯病菌和玉米茎基腐菌的室内毒力。针对高效药剂,以孙云沛法评价各配比的增效作用。结果表明,己唑醇和咯菌腈对小麦纹枯病菌的室内毒力最高,EC_(50)值分别为0.094 mg/L和0.312 mg/L;咪鲜胺和己唑醇对玉米茎基腐病菌的室内毒力最高,EC_(50)值分别为0.088 mg/L和0.593 mg/L。己唑醇和咯菌腈按照不同比例复配对小麦纹枯病菌没有明显的增效作用。咪鲜胺和己唑醇按照1:5、1:2和1:1比例复配对玉米茎基腐病菌均表现出明显的增效效果,其中1:5配比的共毒系数为212.2,增效作用最为显著,生产上可考虑用于上述病害防治。

抗戊唑醇禾谷丝核菌的渗透压敏感性及相对渗率变化研究

经药剂筛选和UV诱导,分别获得了抗戊唑醇的禾谷丝核菌Rhizoctonia cerealis菌株,比较了其与敏感菌株对渗透压的敏感性和经不同浓度戊唑醇处理后的相对渗率变化。在相同的渗透压环境下,敏感菌株对渗透压较抗性菌株更为敏感。不同抗性菌株间对渗透压的敏感性在低渗和中渗环境下差异不大,在高渗环境下有一定的差异;而敏感菌株只对低渗透压(1%葡萄糖)和高渗透压(8%葡萄糖)相对较为敏感。用不同浓度的戊唑醇处理后,与敏感菌株相比,抗性菌株能在较短的时间里(1h)渗出更多的电解质,但最终前者的相对渗率要明显高于后者。用高浓度的戊唑醇处理抗性菌株后,其相对渗率明显高于低浓度处理。

江苏省小麦纹枯病菌致病力研究

江苏省小麦纹枯病菌致病力研究史建荣王裕中沈素文陈怀谷史建荣：男，３４岁，博士研究生，副研究员。本研究系国家“九五”科技攻关、江苏省“九五”重大科技攻关项目的研究内容。收稿日期：１９９６－０７－１５（江苏省农业科学院植物保护研究所，南京２１００１４）关...

小麦纹枯病菌核糖体基因内转录区序列比较

对 7个从江苏省小麦纹枯病样本分离到的丝核菌菌株,进行形态学鉴定、融合群分类和致病性测定,提取病菌的DNA,采用通用引物ITS1(TCCGTAGGTGAACCTGCGG)和ITS4 (TCCTCCGCTTATTGATATGC),扩增病菌的rDNA内转录区(ITS),并对扩增产物进行了测序。用这些序列在NCBI中进行BLAST分析,得到与这些菌株亲缘关系最近的菌株序列,并明确了这些菌株的分类地位。对以上的菌株序列进行Alignment分析,结果表明,病菌的 5. 8SrDNA序列高度保守,而ITS区的可变性则相对较高,在双核和多核丝核菌、双核丝核菌CAG1融合群和非CAG1融合群菌株间存在差异,可用于反映菌株间的进化关系和双核丝核菌种下分类。

小麦种质抗纹枯病性的鉴定和遗传分析

对 2 30 0余份创新种质和引进种质抗纹枯病性进行了自然病地初步鉴定 ,在此基础上选择一批抗性好的种质进行了人工病圃鉴定 ,共评选出高抗纹枯病的创新种质 1 4份 ,引进种质 2 1份。这些种质都兼抗 1～ 3种其它病害 ,且综合性状较好 ,其中创新种质最好 ,为抗纹枯病育种提供了良好的种质材料。还选用 7个抗纹枯病性不同的亲本组配成半双列杂交组合。采用 Hayman法进行了基因效应分析 ,结果表明 ,抗纹枯病性的遗传不符合加性—显性模型。

中国河南省小麦纹枯病菌对苯醚甲环唑及戊唑醇的敏感性

为明确中国河南省小麦纹枯病菌对苯醚甲环唑及戊唑醇的敏感性,采用菌丝生长速率法测定了2种药剂对采自河南省15个地市分离的95株病菌的毒力。结果显示：供试菌株对苯醚甲环唑和戊唑醇的EC_（50）值分别在0.014-1.609和0.002-0.064μg/m L之间。敏感性频率分布图显示：在小麦纹枯病菌群体中已出现对苯醚甲环唑敏感性下降的亚群体,而对戊唑醇则不明显;可将其中呈连续单峰频次分布的大多数菌株群体的平均EC_（50）值（0.165±0.101）和（0.019±0.008）μg/m L分别作为小麦纹枯病菌对苯醚甲环唑和戊唑醇的相对敏感基线。苯醚甲环唑的平均EC_（50）值是戊唑醇的26倍,表明该病原菌对戊唑醇更为敏感。不同地区病原菌群体间对苯醚甲环唑和戊唑醇的敏感性均存在显著性差异。小麦纹枯病菌对2种杀菌剂的敏感性之间存在着微弱的正相关性。本研究结果可为河南省小麦纹枯病防控杀菌剂的合理使用提供理论依据。

小麦品种(系)对纹枯病抗性鉴定及抗病资源的筛选

室内苗期采用菌落上直播催芽麦粒法和田间连续 2年采用小麦粒砂土壤接菌法对 2 5个小麦品种进行了抗纹枯病性鉴定。结果表明 ,品种间抗感性存在明显差异 ,室内和田间抗感性表现基本一致 ,田间两个年度间抗感性表现具有相对的稳定性。品种南阳 82 5 0 5、S0 48、荆麦 6 6等 7个品种室内病级为 1 0 3～ 1 5 0 ,田间相对抗病性指数为 0 40～ 0 79,两个年度均表现为中抗。 1998～ 2 0 0 0年度共鉴定品种材料 5 48份 ,抗性材料占 17 9% ,其中湖北省94 5 0 36、96 95 1等 11份区预试材料表现为中抗 ,占区预试鉴定材料总数的 2 0 8%。

转Rs-AFP2基因小麦的分子分析及其纹枯病抗性

Rs-AFP2属于r-硫堇类抗菌肽,主要通过形成离子通道直接破坏细胞来杀灭病原菌。本研究通过基因枪介导法结合对目标基因的分子检测,证明已将外源Rs-AFP2基因转入小麦推广品种扬麦12中。通过逐株抗纹枯病接种鉴定、PCR、PCR-Southern blot、Southern blot和RT-PCR/荧光定量RT-PCR(Q-RT-PCR)分析,对转Rs-AFP2基因小麦T1至T4代植株跟踪检测。结果表明,Rs-AFP2在转基因小麦中能够稳定遗传,以单拷贝整合到小麦基因组中,遗传方式符合孟德尔遗传规律,并能在转录水平上表达。对转Rs-AFP2基因小麦的抗病性、主要农艺性状以及Rs-AFP2表达活性分析结果表明,与受体扬麦12相比,Rs-AFP2表达活性高的转基因小麦植株对纹枯病抗性有明显提高,其抗病性可以遗传,而主要农艺性状没有明显差异,证明可以利用Rs-AFP2基因和基因工程途径创制抗纹枯病小麦新种质。

抗纹枯病、根腐病的转SN1基因小麦的获得与鉴定

SN1是源于马铃薯的一种抗菌肽,可以抑制多种植物病原菌的生长。小麦纹枯病(主要病原菌为禾谷丝核菌Rhizoctonia cerealis)和根腐病(主要病原菌为平脐蠕孢菌Bipolaris sorokiniana)是小麦的主要土传真菌病害。本研究利用基因工程技术构建了SN1基因的单子叶植物表达载体pA25-SN1,它受玉米泛素(ubiquitin)启动子的控制;采用基因枪法将pA25-SN1转化小麦推广品种扬麦18幼胚愈伤组织4000块,获得203株再生植株,通过PCR检测出阳性植株55株,转化率为1.38%。对外源SN1转基因小麦T0～T2代植株,进行目标基因的PCR、Southern blot、RT-PCR、荧光定量RT-PCR(Q-RT-PCR)分析和小麦纹枯病菌与根腐病菌接种及其抗病性鉴定。结果表明,转入的SN1基因已经整合到转基因小麦的基因组中,能够在转基因小麦中遗传、转录与表达。SN1基因的表达提高了转基因植株对小麦纹枯病和根腐病的抗性,其抗病性可以遗传。

禾谷丝核菌对井冈霉素的抗性风险预测

采用菌丝生长速率法,测定了4个麦区的5个禾谷丝核菌Rhizoctonia cerealis菌株对井冈霉素的抗药性;在含井冈霉素的PDA平板培养基上对禾谷丝核菌进行继代培养,以诱导抗药性菌系;测定了抗性菌系对其他杀菌剂的交互抗性,并比较了抗性和敏感菌系对渗透压的敏感性及药剂处理后两种菌系培养液内还原糖和可溶性蛋白的含量差异。结果表明,田间禾谷丝核菌菌株对井冈霉素分别产生了7.26、7.75、10.46、14.92和23.31倍的抗性。室内继代培养36代,禾谷丝核菌对井冈霉素的抗性达49.24倍,形成了抗井冈霉素菌系。抗井冈霉素菌系对口恶醚唑、福美双、三唑酮、丙环唑、戊唑醇和咯菌腈分别产生了48.58、21.78、17.62、10.95、2.55和1.78倍的交互抗性。抗性菌系在较低和较高渗透压下其生长抑制率均大于敏感菌系,用井冈霉素处理抗性和敏感菌系后,其体内的电解质都严重外渗,且抗性菌系在10h内的外渗量明显大于敏感菌系。