转基因水稻对稻瘟病的抗性研究

 采用苗期初筛、复筛、抗谱测定和田间自然诱发试验等不同鉴定方法,对经分子检测证明已整合有碱性几丁质酶基因和β-1,3-葡聚精酶基因的22个转化系的转基因水稻植株进行稻瘟病抗性鉴定研究,筛选出对稻瘟病的抗性比原种对照七丝软占有明显提高的一系列转基因水稻品系,其中表现高抗的有来自F4-9转化株系的7个品系。高抗材料的R7代品系,经室内抗谱测定及田间病圃试验结果,仍然表现高抗稻瘟病。本研究通过转基因技术,成功地将优质感病品种改良成高抗品系,研究结果证明了利用基因工程手段培育抗病水稻新品种是一个非常有希望的育种途径。     

Mapping quantitative trait Loci for resistance to rice blast

Quantitative trait loci (QTLs) conferring resistance to rice blast, caused by Magnaporthe oryzae, have been under-explored. In the present study, composite interval mapping was used to identify the QTLs that condition resistance to the 6 out of the 12 common races (IB1, IB45, IB49, IB54, IC17, and ID1) of M. oryzae using a recombinant inbred line (RIL) population derived from a cross of the moderately susceptible japonica cultivar Lemont with the moderately resistant indica cultivar Jasmine 85. Disease reactions of 227 F(7) RILs were determined using a category scale of ratings from 0, representing the most resistant, to 5, representing the most susceptible. A total of nine QTLs responsive to different degrees of phenotypic variation ranging from 5.17 to 26.53% were mapped on chromosomes 3, 8, 9, 11, and 12: qBLAST3 at 1.9 centimorgans (cM) to simple sequence repeat (SSR) marker RM282 on chromosome 3 to IB45 accounting for 5.17%; qBLAST8.1 co-segregated with SSR marker RM1148 to IB49 accounting for 6.69%, qBLAST8.2 at 0.1 cM to SSR marker RM72 to IC17 on chromosome 8 accounting for 7.22%; qBLAST9.1 at 0.1 cM to SSR marker RM257 to IB54, qBLAST9.2 at 2.1 cM to SSR marker RM108, and qBLAST9.3 at 0.1 cM to SSR marker RM215 to IC17 on chromosome 9 accounting for 4.64, 7.62, and 4.49%; qBLAST11 at 2.2 cM to SSR marker RM244 to IB45 and IB54 on chromosome 11 accounting for 26.53 and 19.60%; qBLAST12.1 at 0.3 cM to SSR marker OSM89 to IB1 on chromosome 12 accounting for 5.44%; and qBLAST12.2 at 0.3 and 0.1 cM to SSR marker OSM89 to IB49 and ID1 on chromosome 12 accounting for 9.7 and 10.18% of phenotypic variation, respectively. This study demonstrates the usefulness of tagging blast QTLs using physiological races by composite interval mapping.     

Silicon-induced cell wall fortification of rice leaves: a possible cellular mechanism of enhanced host resistance to blast

ABSTRACT Locations of silicon accumulation in rice leaves and its possible association with resistance to rice blast were investigated by electron microscopy and X-ray microanalysis. A blast-susceptible cultivar, Jinmi, and a partially resistant cultivar, Hwaseong, were grown under a hydroponic culture system with modified Yoshida's nutrient solution containing 0, 50, 100, and 200 ppm of silicon. Electron-dense silicon layers were frequently found beneath the cuticle in epidermal cell walls of silicon-treated plants. Increasing levels of silicon were detected in the outer regions of epidermal cell walls. Silicon was present mainly in epidermal cell walls, middle lamellae, and intercellular spaces within subepidermal tissues. Furthermore, silicon was prevalent throughout the leaf surface, with relatively small deposition on stomatal guard cells in silicon-treated plants. Silicon accumulation and epidermal cell wall thickness in leaves were greater in cv. Jinmi than in cv. Hwaseong. However, the thickness ratios of the silicon layers to epidermal cell walls were greater in cv. Hwaseong (53.25 to 93.28%) than in cv. Jinmi (36.58 to 66.54%). Leaf blast severity was lower in cv. Hwaseong than in cv. Jinmi and was significantly reduced in silicon-treated plants of both cultivars. These results suggest that silicon-induced cell wall fortification of rice leaves may be closely associated with enhanced host resistance to blast.     

Suppression of rice blast by phylloplane fungi isolated from rice plants

Rice phylloplane fungi were evaluated for their potential as biocontrol agents for rice blast disease caused by Magnaporthe grisea. A total of 1923 fungal isolates were obtained from rice plants in fields at Ishigaki and Iwama and from potted plants placed in a cedar woods in Iwama as bait. Although 82.9% of isolated fungi could not be identified, species of Epicoccum were the most prevalent among identified isolates. Of the 1923 isolates, 967 were randomly selected for screening against rice leaf blast. Nine isolates (MKP5111B, MKP5112, J2JMR3-2, K2J131-2, I5R3-1, NOP541, K1KM134-1, NOP5112, MKP33222) suppressed the disease when a conidial or hyphal suspension of both the phylloplane fungus and pathogen were simultaneously used to inoculate rice plants cultured in pots in a growth chamber. Five of the isolates originated from potted plants in the woods and four from Ishigaki, a subtropical island. Five (MKP5111B, MKP5112, NOP541, NOP5112, MKP33222) of the nine isolates strongly suppressed conidial germination of M. grisea (0.7%) and formed inhibition zones (3–5mm width) in dual cultures with the pathogen. Methanol extracts from the isolates also inhibited mycelial growth of the pathogen. These results suggest that the five isolates produced antibiotic(s). These five isolates are likely identical or closely related fungal species because the sequence of their ITS regions were 100% similar. ITS sequence analysis also suggested that J2JMR3-2 was associated with a species of Fusarium. Under field conditions, J2JMR3-2 reduced both leaf and panicle blast severity, and three other isolates (MKP5111B, K1KM134-1, K2J131-2) suppressed leaf blast in one of the three experiments.     

水稻空间诱变育种抗稻瘟病研究

粤香占和青华占2个水稻品种的种子经过高空气球搭载后,选育出株型好、分蘖力强,产量提高的空间诱变高代(6代以上)品系.为了解空间诱变品系对稻瘟病的抗性变异情况,采用36个广东省稻瘟病菌不同致病型的代表菌株分别对粤香占和青华占及其空间诱变品系进行抗谱测定.研究结果表明:①空间诱变能使水稻品种产生稻瘟病抗性变异,有的诱变品系比原种抗病,有的则比原种感病.②空间诱变产生的抗病性变异比较复杂,不同品种的抗病性变异不一样.③空间诱变引起的稻瘟病抗性变异有可能是由多个抗病基因引起的.④空间诱变可以获得抗稻瘟病的特异种质.粤香占是感病品种,本研究测定的抗谱为25%,从其空间诱变品系中,获得高抗稻瘟病的2个品系YX-8和YX-9,抗谱达到94.4%.空间诱变抗病品系的穗颈瘟鉴定试验正在进行中.     

3个水稻持久抗瘟性品种的抗性遗传初步研究

用广谱感瘟籼型品种Ｂ４０分别与３个持久抗瘟性籼型品种Ｔｅｔｅｐ、湘资３１５０和谷梅２号杂交。３个组合的Ｆ_１和Ｆ_２及双亲在苗期用Ｚｈ-２-１菌株喷雾接种。结果表明,Ｔｅｔｅｐ、谷梅２号和湘资３１５０均由２对显性基因控制对试验菌株的抗性,其中前２个品种的基因间表现重叠效应,后１个品种的基因间表现抑制效应。     

OsDCL1基因沉默突变体诱导稻瘟病抗性的转录组分析

稻瘟病是严重危害水稻生长的真菌病害,每年给水稻生产带来重大经济损失。与野生型日本晴NPB相比,OsDCL1基因沉默突变体增强了水稻对稻瘟菌的广谱抗病性。为了解水稻OsDCL1-RNAi突变体的抗病机制,我们分析和比较了它和野生型日本晴NPB在稻瘟菌侵染前后转录组水平的变化。响应稻瘟菌侵染的7 129个差异表达基因(DEGs)中,NPB和OsDCL1-RNAi突变体分别有5 382和5 180个基因表达发生了变化,参与生物胁迫反应、信号通路、蛋白代谢和RNA调控4个通路的基因明显被富集。以野生型未受稻瘟菌侵染的基因表达为对照,在OsDCL1-RNAi突变体中共发现1 318个DEGs,且超过70%是上调的,其中很多基因参与了生物胁迫反应(26.9%)和信号通路(11%),上调表达基因中与生物胁迫反应相关的主要为PR基因和细胞壁相关基因。另外,还发现10个茉莉酸相关基因和11个水杨酸相关基因分别在OsDCL1-RNAi突变体中显著下调和上调表达。在OsDCL1-RNAi突变体中还发现WRKY和MYB等一些转录因子家族以及部分受体类激酶被诱导表达。用qRT-PCR方法验证部分差异表达的基因,其结果与DEGs基本一致。OsDCL1-RNAi突变体中转录组变化的分析,为深入了解该突变体抗瘟性增强的机制奠定了基础。     

三种水稻穗瘟抗性鉴定方法的比较

稻瘟病是由稻瘟病菌Magnaporthe oryzcae引起的水稻重要病害[1].在生产上准确地评估水稻品种对稻瘟病的抗性,特别是对穗瘟的抗性,对品种的选用、合理布局及病害预警具有重要的意义.孙国昌等[2]对稻瘟病的离体接种技术进行了研究,包括注射、涂抹、棉花球包穗和喷雾四种方法,但由于各自的某些缺陷,没有得到广泛的应用.     

浙江省水稻新品种(系)对稻瘟病的抗性研究

用4个稻瘟病菌小种对浙江省280份水稻新品种(系)进行稻瘟病抗性分析,结果表明:浙江省水稻新品种(系)对稻瘟病菌具有较好的抗性,籼稻和粳稻对参试菌株的全抗率分别为21.62%和39.05%,说明粳稻具有较籼稻更广的抗谱;浙江省新品种(系)对ZB13小种具有较高的感病率,籼稻和粳稻的感病率分别为69.37%和52.07%。     

Mediation of partial resistance to rice blast through anaerobic induction of ethylene

ABSTRACT The correlation between anaerobic soil conditions and increased resistance to rice blast disease has long been observed without benefit of an adequate explanation. We researched flood depth, dissolved oxygen (DO), and ethylene relative to expression of partial blast resistance in cvs. M-201, Newbonnet, LaGrue, Mars, and Cypress. Cultivar blast index (BI) and flood DO decreased with increasing flood depth. BIs were positively correlated with DO. Total leaf blast lesions were 3.4 and 3.2 times greater in cvs. M-201 and LaGrue growing in a 5.0-mul liter(-1) DO nutrient solution than when growing in a 0.1-mul liter(-1) DO solution. Treatment with 0.25 mM ethephon, which releases ethylene, lowered BIs of Newbonnet, LaGrue, and Cypress growing upland when applied drench, foliar, or foliar-drench. If flooded, BIs of ethephon-treated cultivars were decreased by drench and foliar-drench applications only. BIs of upland plants were unchanged, whereas BIs of analogous flooded plants increased following treatment with 0.31 mM aminoethoxyvinylglycine (AVG), an ethylene biosynthesis inhibitor. We hypothesize that varying anaerobic conditions mediate production of phytohormones, particularly ethylene, which modify expression of inherent partial blast resistance in these rice cultivars.     

杂交稻对稻瘟病和稻白叶枯病的抗性鉴定

对广东省将推广的54份杂交稻组合对稻瘟病和白叶枯病的抗病性进行了鉴定。对稻瘟病的抗病性鉴定表明:抗病组合共49个,占90.7%;其中,高抗(抗性比≥91%)组合30个,占55.6%。对稻白叶枯病的抗病性鉴定表明:没有高抗(HR)和抗(R)的组合,仅有一个杂交稻组合(西胜2175)表现为中抗(mR),仅占1.9%,其余53个组合都表现感病,占98.1%。进一步分析表明:对稻瘟病表现高抗的30个杂交稻组合都不抗白叶枯病,而中抗白叶枯病的杂交稻组合西胜2175对稻瘟病表现为中抗,抗性比为74%。     

Silicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrier

A series of experiments were performed to study the effects of silicon (Si) on rice blast development, H₂O₂ accumulation and lipid peroxidation in a controlled rice—Magnaporthe grisea pathosystem. Rice plants supplied with Si as a single dose immediately after pathogen inoculation (−/+Si) exhibited the same high protection against disease as plants treated continuously with Si for the whole growth period (+/+Si), with disease severity indices of 20.8% and 19.6%, respectively, which were significantly lower than that for the control treatment with no Si supplied (63.7%). A single application of Si to rice plants before inoculation (+/−Si) conferred partial protection (disease severity index of 33.3%) compared with the control treatment. Silicon induced a rapid but transient burst of H₂O₂ at 24 h after inoculation. The addition of Si to rice plants significantly altered the activities of catalase and lipoxygenase and the concentration of malodialdehyde (indicative of lipid peroxidation) in rice plants. We propose that rice plants may respond to Si by increased H₂O₂ accumulation and lipid peroxidation. In turn, these responses are linked to host defence mechanisms such as lignin production, oxidative cross-linking in the cell wall, phytoalexin production, and the hypersensitive reaction. Thus, the mechanisms of Si-stimulated plant disease protection may extend beyond its established role in physically strengthening cell walls.     

部分中国栽培稻资源对稻瘟病的抗性分析

以苗期16个稻瘟病生理小种的抗谱测定以及成株期叶瘟、穗颈瘟鉴定结果为依据,分析了75份中国栽培稻稻瘟病抗性资源的抗瘟性。结果显示,不同抗性品种的抗谱以及对成株期叶瘟和穗颈瘟的抗性反应差异较大。相关分析表明,苗期抗谱测定结果与成株期叶瘟抗性显著相关,而与穗颈瘟,以及叶瘟与穗颈瘟间相关不显著。根据聚类分析结果,抗性资源可分成9个类群,其中以第6类群包含品种最多,抗谱最广(81.3%～100%),并高抗A、B、E、G群生理小种和抗穗颈瘟,可作为水稻稻瘟病抗性育种优先利用的种质资源。     

Ultrastructural and cytochemical aspects of silicon-mediated rice blast resistance

ABSTRACT Although exogenous application of silicon (Si) confers efficient control of rice blast, the probable hypothesis underlying this phenomenon has been confined to that of a mechanical barrier resulting from Si polymerization in planta. However, in this study, we provide the first cytological evidence that Si-mediated resistance to Magnaporthe grisea in rice correlates with specific leaf cell reaction that interfered with the development of the fungus. Accumulation of an amorphous material that stained densely with toluidine blue and reacted positively to osmium tetroxide was a typical feature of cell reaction to infection by M. grisea in samples from Si+ plants. As a result, the extent of fungal colonization was markedly reduced in samples from Si+ plants. In samples from Si- plants, M. grisea grew actively and colonized all leaf tissues. Cytochemi-cal labeling of chitin revealed no difference in the pattern of chitin localization over fungal cell walls of either Si+ or Si- plants at 96 h after inoculation, indicating limited production of chitinases by the rice plant as a mechanism of defense response. On the other hand, the occurrence of empty fungal hyphae, surrounded or trapped in amorphous material, in samples from Si+ plants suggests that phenolic-like compounds or phytoalexins played a primary role in rice defense response against infection by M. grisea. This finding brings new insights into the complex role played by Si in the nature of rice blast resistance.     

黑龙江省水稻新品种（系）抗稻瘟病性鉴定及利用

1998-2009年采用稻瘟病人工接种和自然感病鉴定2种方法,共鉴定黑龙江省水稻新品种（系）485份（次）,鉴定出抗稻瘟病新品种（系）109份,12年间先后推广了‘空育131’、‘绥粳3号’、‘龙粳12’、‘龙粳14’、‘垦稻12’等49个新品种,推广了3个超级稻品种‘龙粳14’、‘龙粳18’、‘龙粳21’,18个新品种年种植面积超过6.67万hm2,获得了较高的经济效益和社会效益。     

寒地水稻新品种(系)抗稻瘟病性鉴定及利用

１９９２～１９９７年通过稻瘟病人工接种和自然感病２种方法,共鉴定寒地水稻新品种（系）６３２份（次）,鉴定出抗稻瘟病新品种（系）５２份,在黑龙江省先后推广普粘七号、藤系１３７等１２个,获得较好社会效益和经济效益。     

利用RNAi 介导的抗病性获得抗2 种花生病毒的转基因烟草

 分别以花生条纹病毒红安株系(PStV-Hongan)外壳蛋白基因(CP),花生矮化病毒轻型株系(PSV-Mi)和花生黄瓜花叶病毒(CMV-CA)复制酶基因2a为模板,通过PCR 方法分别得到PStV-CP 5′ 端,PSV-Mi 和CMV-CA 2a3′ 端150 bp 的片段,3 种片段混合物为模板经PCR 拼接得到450 bp 的片段,此拼接片段通过Gateway 系统重组至植物表达载体pK7GW1WG2,得到含反向重复拼接片段的植物表达载体pK450。冻融法导入根癌农杆菌(Agrobacterium tumefaciens)菌株GV3101 后,叶盘法转化本生烟(Nicotiana benthamiana),经PCR 检测,获得转基因植株。对T1 代转基因植株分别人工接种3 种病毒,66. 7% 的植株表现对PStV 免疫,9% 的植株表现对CMV-CA 的恢复抗性,全部植株对PSV 感病。siRNA 的Northern blot 结果表明,所有转基因烟草植株都含有病毒特异siRNA,siRNA 含量随接种后时间延长而衰减。