稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

江西稻瘟病菌稻巨座壳致病性分化年度动态变化

为明确江西水稻种植区稻巨座壳菌（稻瘟病病菌）的年际变化规律,本研究采用7个中国鉴别寄主与30个单基因鉴别品系两套鉴别寄主分别鉴定分析了2006-2018年间从江西37个水稻主要种植县市分离的1 161个稻瘟病单孢菌的生理小种、致病力、致病类型与无毒基因型等。研究结果表明,江西稻瘟病菌可以分成7群49个生理小种,其中ZA、ZB、ZC群为优势种群,ZB13为优势小种,出现频率为18.00%,以毒性较强的强致病力菌株为主;江西稻瘟病菌在生理小种构成、优势小种、致病力年际变化方面均具有3-5年的周期性;江西历年稻瘟病菌的致病类型较为丰富且存在年度差异,菌株致病类型占各年度总菌株数的82.79%-98.21%,优势致病型菌株占当年总菌株的3.57%-5.77%;历年稻瘟病菌的无毒基因个数为24-29个,其中Avr-Pizt、Avr-Piz5、Avr-Pik、Avr-Pik(C)在历年供试菌株中出现频率较高,说明与之相对应的抗瘟基因在江西抗病育种与抗性品种布局中具有较好的应用价值。     

水稻广谱抗稻瘟病基因研究进展

稻瘟病是水稻生产中的最严重病害之一,由于稻瘟菌小种的高度变异性,垂直抗性基因难以持续控制稻瘟病的危害,因此,克隆和利用广谱持久抗瘟基因被认为是解决稻瘟病问题最经济有效的策略。本文从广谱抗源的筛选与利用,广谱抗瘟基因的定位、克隆与应用等方面对水稻广谱抗稻瘟病基因研究取得的进展进行了概述,并介绍了广谱抗性分子机理的最新研究进展。基于国内外稻瘟病抗性基因研究的现状及趋势,以及我国丰富的抗瘟水稻种质资源,克隆越来越多的广谱抗瘟基因具有重要的理论与应用价值。     

广东地方稻种暹罗占抗稻瘟病基因分析

广谱抗病基因的利用是控制稻瘟病最有效和最经济的方法。来源于华南的地方稻种暹罗占对稻瘟病菌表现出广谱抗性,以普感品种丽江新团黑谷为轮回亲本选育的暹罗占近等基因系NIL-XLZ对测试的44个不同来源稻瘟病菌的抗性频率为84.4%,其抗谱优于广谱抗瘟基因Pi2、Piz,与抗瘟基因Pi9和Pi50相近。为进一步了解暹罗占抗稻瘟病的遗传基础,以感病品种广恢290为母本、暹罗占为父本,构建了广恢290/暹罗占的F2遗传分离群体。选取致病谱较广的稻瘟病菌代表菌株GD08-T19对来源于广恢290/暹罗占的F1与F2个体进行了抗病遗传分析,结果显示F1个体全表现抗病,1760个F2个体的抗感分离比率为4.06∶1,表明暹罗占至少含有一个显性的抗稻瘟病基因。利用分布于Pi2、Pi1、Pita座位附近的44对SSR引物,对构建的抗/感基因池及遗传分离个体进行了分析,将暹罗占含有的一个抗瘟基因定位于水稻第6染色体Pi2/Pi9/Pi50基因家族区域247 kb的范围内。抗菌谱分析、基因特异性分子标记检测及测序分析结果表明:暹罗占含有广谱抗瘟基因Pi50。本研究结果为暹罗占在水稻抗病育种上的应用提供了重要依据。     

粳稻子预44抗LP11稻瘟病菌基因Pizy6(t)的定位

稻瘟病是世界范围内严重威胁水稻(Oryza sativa)生产可持续发展的主要病害之一,每年造成10%–30%的水稻产量损失。抗瘟水稻品种的培育和育种利用是解决稻瘟病危害最经济有效的方法。对新的致病性菌株进行分离和筛选是定位与克隆抗病新基因及抗病育种的基础。选择分离自不同稻瘟病发生重灾区的单孢菌株,对广谱抗瘟水稻子预44和感病水稻江南香糯进行致病性鉴定,筛选出两材料间致病性差异明显的5个菌株;进一步利用子预44、湘资3150、9311、日本晴、丽江新团黑谷、中花11、TP309和江南香糯8个抗瘟性不同的水稻材料,对筛选的菌株进行致病性鉴定。结果显示,LP11能使广谱抗瘟籼稻湘资3150严重发病,推测其很可能是新进化出来的强致病菌株。利用子预44和江南香糯杂交构建的F2群体进行抗性遗传分析,结果表明子预44对LP11菌株的抗性是由单显性基因控制。利用SSR分子标记和图位克隆方法在子预44中定位了1个抗稻瘟病基因Pizy6(t)。研究结果不仅为抗病相关研究提供了有价值的新菌株,而且为子预44中抗稻瘟病基因Pizy6(t)的克隆奠定了基础。     

稻瘟菌无毒基因研究进展

无毒基因编码的产物激发病原物与植物特异性相互作用。水稻与稻瘟菌之间的特异互作符合“基因对基因”关系。从研究稻瘟菌无毒基因的意义、已鉴定和克隆的稻瘟菌无毒基因、稻瘟菌无毒基因与其抗病基因的互作特点等几个方面,对稻瘟菌无毒基因研究进展作了简要评述 。     

稻瘟病菌无毒基因研究进展

水稻与稻瘟病菌之间的特异互作符合基因对基因假说。本文将从稻瘟病菌与水稻抗病基因间的互作特点、稻瘟病菌的分子标记、已克隆的稻瘟病菌无毒基因三个方面对稻瘟病菌无毒基因研究进展作简要介绍     

稻瘟菌无毒基因的分子检测及其指纹类型分析

针对稻瘟菌中Avr-Pita与AvrPiz-t两个无毒基因,设计特异性引物,利用PCR方法对吉林省103株稻瘟菌菌株进行分子检测,并结合AVR-Pib、AVR-C039、PWL2和ACE1 4个无毒基因在其中85个菌株中的分布情况,绘制出基于这6个无毒基因的指纹类型.结果表明,Avr-Pita基因和AvrPiz-t基因在吉林省稻瘟菌中的分布频率分别为86.41%和62.14%;建立的指纹类型显示,85株稻瘟菌分布于9种类型中,其中类型1为主要类型,其分布频率为40.00%:根据30个已知生理小种的菌株在指纹类型中的分布情况,初步发现类型7与吉林省优势生理小种ZE1可能存在对应关系,并应用于稻瘟菌优势生理小种的特异性分子检测;其他指纹类型与中国生理小种并无明显对应关系.明确了无毒基因Avr-Pita与AvrPiz-t在吉林省稻瘟菌中的分布情况,构建了无毒基因的指纹类型,为水稻抗瘟育种和稻瘟菌优势小种的分子监测提供理论依据.     

鸡舍环境中tri5基因阳性镰孢菌的分离及其产毒特性

【目的】探讨利用tri5-PCR鉴定产毒镰孢菌的可行性以及tri5阳性菌株的产毒特性和产毒条件。评估鸡舍空气和固体基质中镰孢菌分离株中产单端孢霉烯族毒素潜力的发生率。【方法】利用编码单端孢霉烯族毒素合成酶的起始基因(tri5基因)对139株镰孢菌分离株进行PCR检测,并对tri5阳性菌株进行产毒培养,通过免疫亲和柱-高效液相色谱方法来检测培养产物中的T-2毒素和HT-2毒素含量。【结果】共筛选到42株tri5阳性菌株,其中分离自鸡舍空气中的10株tri5阳性菌株经产毒培养后7株菌株产生T-2毒素(1.36-5 ng/mL)或HT-2毒素(6.1-17.1 ng/mL)。在5℃-20℃间隔24 h变温、光照与黑暗间隔24 h交替、前期振荡后期静止的培养条件下培养9 d镰孢菌的产毒量最高;镰孢菌的产毒量与温度和时间显著相关,而与菌丝体干重无显著相关性。【结论】与传统鉴定产毒镰孢菌的方法比较,tri5-PCR更适用于快速、准确地检测大量鸡舍环境样本中的产毒镰孢菌。本研究为养殖环境的产毒镰孢菌的危害预警和控制提供技术支撑和理论依据。     

水稻稻瘟病抗性基因的克隆、育种利用及稻瘟菌无毒基因研究进展

稻瘟病是世界上影响水稻(Oryza sativa)粮食生产的主要病害之一, 抗病基因的发掘与利用是抗病育种的基础和核心。随着寄主水稻和病原菌稻瘟病菌(Magnaporthe oryzae)基因组测序和基因注释的完成, 水稻和稻瘟病菌的互作体系成为研究植物与真菌互作的模式系统。该文对稻瘟病抗病基因的遗传、定位、克隆及育种利用进行概述, 并通过生物信息学分析方法, 探讨了水稻全基因组中NBS-LRR类抗病基因在水稻12条染色体上的分布情况, 同时对稻瘟病菌无毒基因的鉴定及无毒蛋白与抗病蛋白的互作进行初步分析。最后对稻瘟病抗病基因研究存在的问题进行分析并展望了未来的研究方向, 以期为水稻抗稻瘟病育种发展和抗病机制的深入理解提供参考。     

Overexpression of BAS1 in rice blast fungus can promote blast fungus growth,sporulation and virulence in planta

Background: BAS1 is biotrophy-associated secreted protein of rice blast strain (Magnaporthe oryzae). In order to study the effect of BAS1 on virulence of rice blast strain, we characterized function of BAS1 using a purified prokaryotic expression product of BAS1 and its overexpression strain. Results: Our results showed in vitro the purified expression product caused rapid callose deposition and ROS production in rice leaves and calli, indicated it triggered transient basal defense. When the purified expression product of BAS1 was sprayed onto rice leaves, and 24 h later the leaves were inoculated with blast strain, the results showed the size and number of lesions, on purified BAS1 product-pretreated leaves of the Lijiangxintuanheigu (LTH) challenged with blast strain, was higher than those in BAS1-untreated leaves directly challenged with the same strain, which suggested the defense response trigged by BAS1 can be overcome by other effectors of the fungus. More severe symptoms, higher sporulation, higher relative fungal growth and more lower expression level of defense-related genes appeared in LTH leaves challenged with overexpression strain 35S:BAS1/Mo-2 than those in LTH inoculated with wild-type strain. Conclusions: These data suggest both in vitro pretreatment with BAS1 prokaryotic expression products and overexpression in blast strains can increase virulence of blast fungus.     

Three types of defense-responsive genes are involved in resistance to bacterial blight and fungal blast diseases in rice

Bacterial blight and fungal blast diseases of rice, caused by Xanthomonas oryzae pv. oryzae and Pyricularia grisea Sacc., respectively, are two of the most devastating diseases in rice worldwide. To study the defense responses to infection with each of these pathogens, expression profiling of 12 defense-responsive genes was performed using near-isogenic rice lines that are resistant or susceptible to bacterial blight and fungal blast, respectively, and rice cultivars that are resistant or susceptible to both pathogens. All 12 genes showed constitutive expression, but expression levels increased in response to infection. Based on their expression patterns in 12 host-pathogen combinations, these genes could be classified into three types, pathogen non-specific (6), pathogen specific but race non-specific (4) and race specific (2). Most of the 12 genes were only responsive during incompatible interactions. These results suggest that bacterial blight and fungal blast resistances share common pathway(s), but are also regulated by different defense pathways in rice. Activation of the corresponding R gene is the key step that initiates the action of these genes in defense responses. The chromosomal locations and pathogen specificities of seven of the 12 genes were consistent with those of previously identified quantitative trait loci for rice disease resistance, which indicates that some of the 12 genes studied may have a phenotypic impact on disease resistance in rice.     

Physiological and cytological mechanisms of silicon-induced resistance in rice against blast disease

Rice (Oryza sativa L.) blast disease caused by Magnaporthe grisea is one of the most destructive diseases in the rice-growing areas of the world. Silicon is an important nutritional element especially for rice. Two near-isogenic lines of rice with different resistance to blast disease, i.e. CO39 (susceptible) and C101LAC (Pi-1) (resistant), were selected to determine the effects of Si amendment on the severity and incidence of rice blast disease. The physiological and cytological mechanisms involved in the induced disease resistance by silicon were investigated. Exogenous Si application at a concentration of 2 mM reduced the disease index by 45% for CO39 and 56% for C101LAC (Pi-1). Si application alone did not change lignin content and the activities of defense-related enzymes including peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) in rice leaves of both isogenic lines. However, after inoculation with M. grisea, Si-treated rice plants significantly increased the activities of POD, PPO and PAL in leaves of both isogenic lines. Si and lignin content were also significantly increased in Si-treated inoculated seedlings. Environmental scanning electron microscope observations revealed that Si amendment resulted in higher Si deposit on dumbbell bodies in the rice leaves and silicon papilla accumulation on the guard cell of stoma. These results suggest that silicon-induced defense response and cell silicification of rice leaves altogether contribute to the silicon-induced rice resistance to blast disease.     

Tagging genes for blast resistance in rice via linkage to RFLP markers

Summary Both Pi-2(t) and Pi-4(t) genes of rice confer complete resistance to the blast fungal pathogen Pyricularia oryzae Cav. As economically important plant genes, they have been recently characterized phenotypically, yet nothing is known about their classical linkage associations and gene products. We report here the isolation of DNA markers closely linked to these blast resistance genes in rice. The DNA markers were identified by testing 142 mapped rice genomic clones as hybridization probes against Southern blots, consisting of DNA from pairs of nearly isogenic lines (NILs) with or without the target genes. Chromosomal segments introgressed from donor genomes were distinguished by restriction fragment length polymorphisms (RFLPs) between the NILs. Linkage associations of the clones with Pi-2(t) and Pi4(t) were verified using F₃ segregating populations of known blast reaction. Cosegregation of the resistant genotype and donor-derived allele indicated the presence of linkage between the DNA marker and a blast resistance gene. RFLP analysis showed that Pi-2(t) is closely linked to a single-copy DNA clone RG64 on chromosome 6, with a distance of 2.8+1.4(SE) cMorgans. Another blast resistance gene, Pi-4(t), is 15.3+4.2(SE) cMorgans away from a DNA clone RG869 on chromosome 12. These chromosomal regions can now be examined with additional markers to define the precise locations of Pi-2(t) and Pi-4(t). Tightly linked DNA markers may facilitate early selection for blast resistance genes in breeding programs. These markers may also be useful to map new genes for resistance to blast isolates. They may ultimately lead to the cloning of those genes via chromosome walking. The gene tagging approach demonstrated in this paper may apply to other genes of interest for both monogenic and polygenic traits.     

Development of pre-isogenic lines for rice blast-resistance by marker-aided selection from a recombinant inbred population

To increase the available set of near-isogenic lines (NILs) for blast-resistance in rice, we have developed a general method for establishing NILs from populations of fixed recombinants that have been used for gene mapping. We demonstrated the application of this method by the selection of lines carrying genes from the rice cultivar Moroberekan. Moroberekan is a West African japonica cultivar that is considered to have durable resistance to rice blast. Multiple genes from Moroberekan conferring complete and partial resistance to blast have previously been mapped using a recombinant inbred (RI) population derived from a cross between Moroberekan and the highly and broadly susceptible indica cultivar CO39. To analyze individual blast-resistance genes, it is desirable to transfer them individually into a susceptible genetic background. This RI population, and the associated data sets on blast reaction and restriction fragment length polymorphism (RFLP) genotypes, were used for selection of lines likely to carry individual blast-resistance genes and a minimum number of chromosomal segments from Moroberekan. Because skewed segregation in the RI population favored CO39 (indica) alleles, resistant lines carrying 8.7–17.5% of Moroberekan alleles (the proportion expected after two or three backcrosses) could be selected. We chose three RI lines carrying different complete resistance genes to blast and two RI lines carrying partial resistance genes to blast as potential parents for the development of NILs. These lines were subjected to genetic analysis, which allowed clarification of some issues that could not be resolved during the initial gene-mapping study.     

稻瘟病分子生物学研究进展

稻瘟病分子生物学发展迅速,已分子标记定位的稻瘟病主效抗性基因１５个,微效抗性基因３个;水稻抗稻瘟病基因Ｐｉ－ｔａ和Ｐｉ－ｂ已成功克隆。稻瘟病菌系谱与致病型关系可分为简单与复杂两种类型。本文对水稻抗稻瘟病基因的定位和克隆,稻瘟病菌群体遗传结构,致病性遗传、基因组分析、无毒基因克隆、准性生殖等研究进展进行了评述。     

水稻抗稻瘟病天然免疫机制及抗病育种新策略

稻瘟病是水稻最严重的病害之一,由子囊菌(Magnaporthe oryzae)引起。利用抗病品种是防治稻瘟病最经济、最有效的措施。近年来,稻瘟病已发展为研究植物与病原真菌分子互作机制的模式系统,在水稻与稻瘟菌互作和寄主抗性分子生物学、基因组学和蛋白组学等领域取得了一系列重要的研究成果。文章综述了近年来水稻抗稻瘟病两种天然免疫机制,即病原菌相关分子模式诱导和效应蛋白诱导的抗病机制研究的最新进展,讨论了GWAS、TALLEN、CRISPR和HIGS等基因组研究新方法和新技术在水稻抗病育种中的应用,并对目前稻瘟病抗性机制研究和抗病育种中的问题和挑战进行了探讨和展望。     

Creation of blast disease-resistant rice varieties with modern DNA-markers

Based on modern technologies of molecular DNA-markers, blast disease–resistance genes (Pi-ta, Pi-b, Pi-1, Pi-2, and Pi-33) were introgressed and pyramided into domestic rice varieties to give them longterm disease resistance. For that purpose, this case study uses SSR-markers closely linked to these genes, as well as intragenic markers of genes Pi-ta and Pi-b. Multiplex PCR systems were created for simultaneous identification of two resistance genes in the hybrid progeny for the following combinations: Pi-1 + Pi-2, Pi-ta + Pi-b, Pi-ta + Pi-33.     

Identification of RAPD markers linked to a major blast resistance gene in rice

Rice blast, caused byPyricularia grisea, is a major production constraint in many parts of the world. The Korean rice variety Tongil showed high levels of resistance for about six years when widely planted under highly disease-conducive conditions, before becoming susceptible. Tongil was found to carry a single dominant gene, designatedPi-10t, conferring resistance to isolate 106 of the blast pathogen from the Philippines. We report here the use of bulked segregant RAPD analysis for rapid identification of DNA markers linked toPi-10t. Pooled DNA extracts from five homozygous blast-resistant (RR) and five susceptible (rr) BC₃F₂ plants, derived from a CO39 × Tongil cross, were analyzed by RFLP using 83 polymorphic probes and by RAPD using 468 random oligomers. We identified two RAPD markers linked to thePi-10t locus: RRF6 (3.8 ± 1.2 cM) and RRH18 (2.9 ± 0.9 cM). Linkage of these markers withPi-10t was verified using an F2 population segregating forPi-10t. The two linked RAPD markers mapped 7 cM apart on chromosome 5. Chromosomal regions surrounding thePi-10t gene were examined with additional RFLP markers to define the segment introgressed from the donor genome.Pi-10t is likely to be a new blast-resistance locus, because no other known resistance gene has been mapped on chromosome 5. These tightly linked RAPD markers could facilitate early selection of thePi-10t locus in rice breeding programmes.     

Identification of five new blast resistance genes in the highly blast-resistant rice variety IR64 using a QTL mapping strategy

Rice progenies used for the construction of genetic maps permit exhaustive identification and characterization of resistance genes present in their parental cultivars. We inoculated a rice progeny derived from the cross IR64 x Azucena with different Magnaporthe grisea isolates that showed differential responses on the parental cultivars. By QTL mapping, nine unlinked loci conferring resistance to each isolate were identified and named Pi-24( t) to Pi-32( t). They could correspond to nine specific resistance genes. Five of these resistance loci (RLs) were mapped at chromosomal locations where no resistance gene was previously reported, defining new resistance genes. Using degenerate primers of the NBS (nucleotide binding site) motif found in many resistance genes, two resistance gene analogues (RGAs) IR86 and IR14 were identified and mapped closely to two blast RLs (resistance identified in this study, i.e. Pi-29(t) and Pi-30(t) respectively). These two RLs may correspond to the Pi-11 and Pi-a blast resistance genes previously identified. Moreover, the ir86 and ir14 genes have been identified "in silico" on the indica rice cultivar 93-11, recently sequenced by Chinese researchers. Both genes encodes NBS-LRR-like proteins that are characteristics of plant-disease resistance genes.