抗褐飞虱水稻品种的培育及其抗性表现

褐飞虱Nilaparvata lugens(Stl)是危害水稻的主要虫害之一,发掘和利用新的抗褐飞虱基因培育抗性品种是目前防治褐飞虱最经济有效的方法之一。抗褐飞虱基因来自药用野生稻的抗虫品种B5,对褐飞虱生物型1和2具有高度抗性,B5携带的抗性基因Bph14被定位在第3染色体上。本研究以B5-10为抗源,以优良杂交稻亲本扬稻6号为受体亲本,通过复交和回交,利用与Bph14紧密连锁的分子标记MRG2329在后代中进行分子标记辅助选择,通过苗期分子标记检测和成株期农艺性状选择,最后育成恢复系R476和杂交组合广两优476。采用苗期群体鉴定技术对R476和广两优476的褐飞虱抗性进行了鉴定,R476和广两优476的抗性水平分别为中抗和中感。广两优476在稻飞虱发生较重的稻田进行试种示范,与对照品种扬两优6号和两优培九相比,广两优476对稻飞虱表现出明显的抗性。研究结果表明在育种过程中利用分子标记辅助选择Bph14基因是培育抗褐飞虱水稻品种的有效途径之一。     

不同水稻品种对褐飞虱的抗性评价

【目的】调查目前江西主栽水稻品种对褐飞虱Nilaparvata lugens(St?l)的抗性级别,为抗褐飞虱水稻品种的选育、推广和褐飞虱的防治提供基础数据。【方法】采用标准苗期集团筛选法,评价了江西推广的25个水稻品种(3个早稻品种,15个中稻品种,7个晚稻品种)苗期和成株期对褐飞虱的抗性。【结果】早稻品种苗期和成株期的抗级为7级(感虫)或9级(高感);中稻品种中隆两优534和五山丝苗苗期抗级为5级(中抗),但成株期抗性丧失,其余品种苗期、成株期的抗性等级为7级(感虫)或9级(高感);晚稻品种高优红88和丰源优2297苗期抗级分别为7级(感虫)和9级(高感),成株期抗级均为5级(中抗),其余品种苗期、成株期的抗级为7级(感虫)或9级(高感)。【结论】目前在江西普遍种植的水稻品种中,多为感虫和高感品种,抗性品种较少,且抗性不高。     

水稻褐飞虱综合治理研究与示范——农业公益性行业专项“水稻褐飞虱综合防控技术研究”进展

针对我国褐飞虱Nilaparvata lugens(Stl)近年来严重为害及其对当家农药品种抗性急剧上升的现状,本项目在华中、华南、华东地区等代表性地区开展褐飞虱灾变规律、抗虫品种培育、抗药性监测及复配农药新剂型开发、生态治理新技术研究、预测预报技术、可持续治理技术集成与示范研究。结果表明:越南、老挝等境外虫源地及我国华南、长江中下游等地田间褐飞虱种群仍以2型为主,3型次之。田间小气候是褐飞虱逃避高温的关键因素,褐飞虱在上午气温升高时大量向温度较低的水稻基部20cm范围内转移以逃避高温,将高温天气造成的不利影响降至最低。不同栽插方式对褐飞虱发生量有明显影响,手栽稻田褐飞虱发生最重,机插稻次之,直播稻最轻。不同水稻品种、N肥施用水平对褐飞虱的发生有明显的影响:超级杂交稻褐飞虱虫口密度明显高于常规杂交稻,高N肥施用量促进褐飞虱的发生,且水稻品种与N肥施用量对褐飞虱发生量的影响有明显交互作用。抗药性监测结果表明:我国褐飞虱对吡虫啉有极高水平抗性(168.1～561.5倍),对噻嗪酮为低到中等水平抗性(4.2～33.1倍),对氟虫腈为敏感性降低到高水平抗性(2.7～67.7倍),对烯啶虫胺与毒死蜱为低水平抗性。筛选出噻虫嗪、吡蚜酮、唏啶虫胺和仲丁威4种高效低毒的防治单剂。研制出3种农药复配新制剂,其中1种新制剂已获得农业部药检所正式登记,且规模化生产,2种新制剂已进入农药登记程序。精细定位水稻抗褐飞虱基因Bph6、Bph7、Bph9、Bph15、BG1222,并找到了它们的近距离共分离分子标记。培育出高产、优质、熟期适宜、含有抗稻飞虱基因Bph14的水稻新品种广两优476。储备了一批聚合多抗稻飞虱基因的水稻亲本材料。研发出在田埂种植芝麻、大豆等天敌诱集植物,增加褐飞虱卵期寄生蜂和捕食性天敌蜘蛛的种群数量的轻简化生态调控技术;研发出对褐飞虱成、若虫取食及雌成虫产卵均有驱避作用的植物提取物混配剂3种;研发出显著提高稻虱缨小蜂对田间褐飞虱卵寄生率的引诱剂1种。建立了褐飞虱环境友好防控技术集成体系,并在湖南、湖北、广东、广西、浙江、江西6省区示范应用,取得了良好的经济、生态、社会效益。     

利用回交重组自交群体检测水稻抗褐飞虱数量性状基因座

利用由98个系组成的Nipponbare/Kasalath//Nipponbare回交重组自交系（Backcross Inbred lines,BILs）作图群体（BC1F9）,进行水稻抗褐飞虱数量性状基因座（Quantative Trait Locus,QTL）的检测和遗传效应分析。采用苗期集团鉴定方法,并以死苗率作为抗褐飞虱表型值,分析亲本和98个BILs抗褐飞虱表现。共检测到3个苗期抗褐飞虱QTL,分别位于2、10和12染色体上,各QTL的LOD值为2.01～2.33,贡献率为10.4%～16.6%,可解释群体总表型变异的39.0%。这3个数量性状基因座对褐飞虱的抗性均来自抗虫亲本Kasalath。与这些数量性状基因座连锁的分子标记可望应用于聚合多个抗性基因,培育对褐飞虱具有水平抗性水稻新品种的育种实践中。     

水稻抗褐飞虱基因Bph3和Bph24(t)的聚合育种利用

在水稻(Oryza sativa)的生产实践中,常常会受到褐飞虱(Nilaparvatal lugens)等多种病虫害的威胁。聚合不同抗性基因,培育抗性品系,是应对各种生物胁迫最有效的策略。传统香稻由于其自身抗性等条件限制,无法大面积推广。本研究结合分子标记辅助选择(marker-assisted selection, MAS)技术,以含有抗褐飞虱基因Bph3或Bph24(t)的供体亲本与含有抗稻瘟病基因(Pi2、Pib或Pimh)、抗白叶枯病基因(Xa23)或香味基因(badh2)的优质三系杂交水稻保持系和恢复系进行多亲本复合杂交。将抗褐飞虱基因聚合到优良水稻品系中,筛选获得41个含有抗褐飞虱基因且与其他目的基因以不同组合方式相聚合的稳定品系。抗性鉴定、香味检测和农艺性状测定表明,各聚合品系的褐飞虱抗性水平较受体亲本均有明显提升,且具有双抗、三抗和/或香味等优良表型。这些新品系为多抗、优质水稻新品种的选育提供新的种质材料。     

二代褐飞虱对粳稻危害损失及防治指标研究

不同水稻生育期．褐飞虱的不同虫口密度的增长倍数不同;同一生育期．以低密度的增殖倍数大于高密度．但总虫量仍以高密度处理极显地高于低密度处理。水稻产量损失与二代褐飞虱百丛虫量间呈显正相关(r=0．9557^ ),两关系式为：Y=-16．3732 9．437tg(x);防治指标为150-200头／百丛。当二代楫飞虱早发、中等以上发生程度时．必须施药防治。     

捕食稻飞虱卵的天敌——黑肩绿盲蝽

黑肩绿盲蝽Cyrtorrhinus lividipennis Reuter是稻褐飞虱的重要天敌之一。1976年我们配合阳江县海陵公社进行以保护天敌为中心的工作时,经田间调查,早稻后期黑肩绿盲蝽的密度达63头/百丛(最高达243头/百丛),被捕食的稻褐飞虱卵粒达19.2％;晚稻后期,黑肩绿盲蝽的密度为543头/百丛(最高达681头/百丛),比稻褐飞虱密度高出数倍,捕食稻褐飞虱卵粒达74.23％。     

中晚粳稻区褐飞虱防治策略的研究

为了解二代褐飞虱对粳稻的为害损失,确定合理的防治指标和防治策略,为科学使用噻嗪酮、吡虫啉等药剂防治稻飞虱提供依据,作者在武育粳３号上,通过接不同的虫量,并进行笼罩的方法,研究了二代褐飞虱的增殖规律和为害损失及防治指标。并进行了锐劲特防治不同世代褐飞虱策略试验。结果表明,不同水稻生育期,不同虫口密度的褐飞虱的增长速度不一样;在水稻同一生育期,低密度的增殖速度要大于高密度,但总虫量高密度区显著高于低密度区。二代褐飞虱百丛虫量与水稻产量损失呈极显著正相关（ ｒ＝ ０．９５７６**）,关系式为 ｙ＝－ １８．３３０２＋ １０． ２８９２ｌｇｘ（ｘ为 ８月上旬褐飞虱百丛虫量）,防治指标为 １１０－１２０头。因此,在早发年,迁入量较大的年份必须采取“治二压三控四”的策略,１９９６－１９９７年全面推广这一策略,成效显著。     

褐飞虱侵害对不同抗性水平水稻根部吸收氮、磷、钾的影响

为了解褐飞虱Nilaparvata lugens侵害后水稻抗性水平与根系吸收氮（N）、磷（P）、钾（K）的关系,对不同抗性水平的水稻受褐飞虱侵害后的平均受害水平、植株功能损失指数及水稻吸收水培液营养元素N,P,K情况进行了研究。结果表明：不同水稻品种（TN1、协优63、协优963和超级培矮64S/E32）接种褐飞虱后,TN1受害最严重,协优63和超级培矮64S/E32 次之;受害最轻的是协优963,30头/株侵害后植株功能损失指数仅为0.661。40,60和80头/株侵害水稻后根系对K吸收下降程度最显著,其次为P,最后为N;且随褐飞虱侵害时间的延长（6 d, 9 d）影响愈显著。60头/株侵害TN1、协优63、协优963和超级培矮64S/E32后9 d,根系对K吸收下降率分别为164.11%,74.61%,55.16%和46.60%。由此可见,随水稻品种抗性水平的下降,接种褐飞虱后,水稻根部对水培液营养元素吸收下降程度愈显著。本研究结果可对深入阐明不同抗性水平水稻抗（耐）虫机制与根系吸收能力关系提供参考。     

抗褐飞虱基因的发掘、鉴定与利用

水稻是我国最重要的粮食作物,而褐飞虱是水稻生产中的主要害虫,培育与利用抗褐飞虱的水稻品种是综合防治褐飞虱的基础。现从水稻抗褐飞虱种质资源、水稻抗褐飞虱基因的定位与克隆、水稻抗褐飞虱的分子机理,以及抗褐飞虱水稻的培育等方面,综述水稻抗褐飞虱基因研究的最新成果。     

Pyramiding and evaluation of the brown planthopper resistance genes Bph14 and Bph15 in hybrid rice

The brown planthopper (BPH) is the most devastating insect pest in rice-producing areas. Shanyou 63 has become a widely cultivated hybrid in China over the last two decades; however, this line has become increasingly susceptible to bacterial blight (BB), blast, and BPH, resulting in a rapid decline in its use in rice production. In this study, a molecular marker-assisted selection (MAS) introgression of Bph14 and Bph15 was performed to improve the BPH resistance of Minghui 63 and its derived hybrids such as Shanyou 63. The effect of pyramiding genes was then comprehensively evaluated using three tests that comprised seedbox screening, feeding rate, and antixenosis for settling in the field. The results showed that the improved hybrids containing a single BPH resistance gene showed enhanced resistance (lower resistance score, honeydew weight and number of BPH settling) compared to conventional hybrids, while pyramiding two genes provided even higher resistance. Moreover, both Bph14 and Bph15 are partial dominance genes, and have a strong dosage effect on the resistance to BPH in the hybrid background, which is useful for breeding BPH-resistant hybrids. Field trial data demonstrated that yields of improved hybrid rice were higher than or similar to the control (Shanyou 63) under natural field conditions. These improved versions could be used in breeding programs for “green super rice.”     

水稻OsLecRK1基因介导的抗褐飞虱功能分析

褐飞虱Nilaparvata lugens St(a)l是对水稻最具破坏性的害虫之一,OsLecRK1是水稻Bph3基因簇中对褐飞虱抗性贡献最大的基因.本文对RHTd(含Bph3)等材料进行了褐飞虱抗性评价,克隆并构建了OsLecRK1过量表达突变体水稻,利用该突变体分析了OsLecRK1基因对褐飞虱若虫存活率、若虫发育历期等生物学参数的影响.结果 表明,含Bph3基因水稻RHTd对褐飞虱的抗性明显地强于含Bph1基因水稻Mudgo和bph2基因水稻ASD7,RHTd水稻的褐飞虱受害指数仅为Mudgo和ASD7水稻的53.5％和24.1％.过量表达OsLecRK1基因能显著地增加水稻对褐飞虱的驱避性和抗生性,褐飞虱雌成虫偏好于在野生型水稻上产卵;突变体水稻上的褐飞虱若虫存活率显著地降低,仅为野生型水稻上若虫存活率的75.2％ ～81.8％,且若虫发育历期显著地延长,羽化率和初羽化雌成虫体重均显著地降低;此外,褐飞虱在突变体水稻上取食分泌的蜜露量只有野生型上的40.3％ ～ 60.9％,褐飞虱单雌产卵量只为野生型51％ ～61.2％,卵孵化率只有野生型的52.2％～56.7％,均显著地减少.结果 表明,含Bph3基因水稻RHTd对褐飞虱的抗性明显地高于分别含Bph1、bph2的水稻Mudgo和ASD7;水稻Bph3基因座的OsLecRK1单个基因过量表达即可显著增加水稻对褐飞虱的抗性,OsLecRK1协同影响褐飞虱的多个生物学参数降低褐飞虱的适合度.     

Insights into the structure–function relationship of brown plant hopper resistance protein,Bph14 of rice plant: a computational structural biology approach

Brown plant hopper (BPH) is one of the major destructive insect pests of rice, causing severe yield loss. Thirty-two BPH resistance genes have been identified in cultivated and wild species of rice Although, molecular mechanism of rice plant resistance against BPH studied through map-based cloning, due to non-existence of NMR/crystal structures of Bph14 protein, recognition of leucine-rich repeat (LRR) domain and its interaction with different ligands are poorly understood. Thus, in the present study, in silico approach was adopted to predict three-dimensional structure of LRR domain of Bph14 using comparative modelling approach followed by interaction study with jasmonic and salicylic acids. LRR domain along with LRR-jasmonic and salicylic acid complexes were subjected to dynamic simulation using GROMACS, individually, for energy minimisation and refinement of the structure. Final binding energy of jasmonic and salicylic acid with LRR domain was calculated using MM/PBSA. Free-energy landscape analysis revealed that overall stability of LRR domain of Bph14 is not much affected after forming complex with jasmonic and salicylic acid. MM/PBSA analysis revealed that binding affinities of LRR domain towards salicylic acid is higher as compared to jasmonic acid. Interaction study of LRR domain with salicylic acid and jasmonic acid reveals that THR987 of LRR form hydrogen bond with both complexes. Thus, THR987 plays active role in the Bph14 and phytochemical interaction for inducing resistance in rice plant against BPH. In future, Bph14 gene and phytochemicals could be used in BPH management and development of novel resistant varieties for increasing rice yield.     

Genetic mapping of the rice resistance-breaking gene of the brown planthopper Nilaparvata lugens

Host plant resistance has been widely used for controlling the major rice pest brown planthopper (BPH, Nilaparvata lugens). However, adaptation of the wild BPH population to resistance limits the effective use of resistant rice varieties. Quantitative trait locus (QTL) analysis was conducted to identify resistance-breaking genes against the anti-feeding mechanism mediated by the rice resistance gene Bph1. QTL analysis in iso-female BPH lines with single-nucleotide polymorphism (SNP) markers detected a single region on the 10th linkage group responsible for the virulence. The QTL explained from 57 to 84% of the total phenotypic variation. Bulked segregant analysis with next-generation sequencing in F₂ progenies identified five SNPs genetically linked to the virulence. These analyses showed that virulence to Bph1 was controlled by a single recessive gene. In contrast to previous studies, the gene-for-gene relationship between the major resistance gene Bph1 and virulence gene of BPH was confirmed. Identified markers are available for map-based cloning of the major gene controlling BPH virulence to rice resistance.     

Marker-assisted pyramiding of brown planthopper (Nilaparvata lugens Stål) resistance genes Bph1 and Bph2 on rice chromosome 12

Brown planthopper (BPH) (Nilaparvata lugens St?l) is a significant insect pest of rice (Oryza sativa L.). We constructed a gene-pyramided japonica line, in which two BPH resistance genes Bph1 and Bph2 on the long arm of chromosome 12 independently derived from two indica resistance lines were combined through the recombinant selection. The gene-pyramiding was achieved based on the previously constructed high-resolution linkage maps of the two genes. Two co-dominant and four dominant PCR-based markers flanking the loci were used to select for a homozygous recombinant line in a segregating population that was derived from a cross between the parental homozygous single-gene introgression lines. BPH bioassay showed that the resistance level of the pyramided line was equivalent to that of the Bph1-single introgression line, which showed a higher level of resistance than the Bph2-single introgression line. The pyramid line should provide a useful experimental means for studying the fine structure of the chromosomal region covering these two major BPH resistance genes.     

Mapping of a broad-spectrum brown planthopper resistance gene, <Emphasis Type="Italic">Bph3</Emphasis>, on rice chromosome 6

The brown planthopper (BPH) is one of the most destructive insect pests of rice in Thailand. We performed a cluster analysis that revealed the existence of four groups corresponding to the variation of virulence against BPH resistance genes in 45 BPH populations collected in Thailand. Rice cultivars Rathu Heenati and PTB33, which carry Bph3, showed a broad-spectrum resistance against all BPH populations used in this study. The resistant gene Bph3 has been extensively studied and used in rice breeding programs against BPH; however, the chromosomal location of Bph3 in the rice genome has not yet been determined. In this study, a simple sequence repeat (SSR) analysis was performed to identify and localize the Bph3 gene derived from cvs. Rathu Heenati and PTB33. For mapping of the Bph3 locus, we developed two backcross populations, BC₁F₂ and BC₃F₂, from crosses of PTB33 × RD6 and Rathu Heenati × KDML105, respectively, and evaluated these for BPH resistance. Thirty-six polymorphic SSR markers on chromosomes 4, 6 and 10 were used to survey 15 resistant (R) and 15 susceptible (S) individuals from the backcross populations. One SSR marker, RM190, on chromosome 6 was associated with resistance and susceptibility in both backcross populations. Additional SSR markers surrounding the RM190 locus were also examined to define the location of Bph3. Based on the linkage analysis of 208 BC₁F₂ and 333 BC₃F₂ individuals, we were able to map the Bph3 locus between two flanking SSR markers, RM589 and RM588, on the short arm of chromosome 6 within 0.9 and 1.4 cM, respectively. This study confirms both the location of Bph3 and the allelic relationship between Bph3 and bph4 on chromosome 6 that have been previously reported. The tightly linked SSR markers will facilitate marker-assisted gene pyramiding and provide the basis for map-based cloning of the resistant gene.     

High-resolution mapping of the brown planthopper resistance gene Bph6 in rice and characterizing its resistance in the 9311 and Nipponbare near isogenic backgrounds

Brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most destructive insect pests of rice. Exploring resistance genes from diverse germplasms and incorporating them into cultivated varieties are critical for controlling this insect. The rice variety Swarnalata was reported to carry a resistance gene (designated Bph6), which has not yet been assigned to a chromosome location and the resistance mechanism is still unknown. In this study, we identified and mapped this gene using the F₂ and backcrossing populations and characterized its resistance in indica 9311 and japonica Nipponbare using near isogenic lines (NILs). In analysis of 9311/Swarnalata F₂ population, the Bph6 gene was located on the long arm of chromosome 4 between the SSR markers RM6997 and RM5742. The gene was further mapped precisely to a 25-kb region delimited between the STS markers Y19 and Y9; and the distance between these markers is 25-kb in Nipponbare genome. The Bph6 explained 77.5% of the phenotypic variance of BPH resistance in F₂ population and 84.9% in BC₂F₂ population. Allele from Swarnalata significantly increased resistance to the BPH, resulted in a reduced damage score. In characterization of Bph6-mediated resistance, the BPH insects showed significant preference between NIL-9311 and 9311 in 3 h and between NIL-NIP and Nipponbare in 120 h after release. BPH growth and development were inhibited, and the insect’s survival rates were lower on Bph6-NIL plants, compared with the parents 9311 and Nipponbare. The results indicate that the Bph6 exerted prolonged antixenotic and antibiotic effects in Bph6-NIL plants, and NIL-9311 plants showed a quicker and stronger effect toward BPH than NIL-NIP plants.     

云南野生稻抗褐飞虱评价及其抗性基因鉴定

褐飞虱是水稻生产中最严重的害虫之一,从野生稻中发掘抗虫基因,有利于培育具有抗虫能力强的水稻新品种。该研究通过对云南野生稻进行温室和大田抗虫鉴定以及9个已知抗褐飞虱基因的PCR鉴定,发现云南野生稻对褐飞虱表现出不同程度的抗性,尤其疣粒野生稻和药用野生稻对褐飞虱表现出高抗,可作为抗虫基因发掘的优良抗源材料;不同褐飞虱抗性的云南野生稻中含有的抗褐飞虱基因差异很大,3种野生稻中均不含Bph1和Bph18(t)抗病基因,景洪普通野生稻和元江普通野生稻可能含bph2基因,东乡普通野生稻可能含bph2、Bph15和Bph27(t)基因,疣粒野生稻中可能含bph2和bph19(t)基因,药用野生稻和药用野生稻(宽叶型)中可能含bph2和Bph6基因,药用野生稻F1中可能含bph2、Bph14和bph20(t)基因,药用野生稻F2中可能含bph2和Bph27(t)基因或者其同源基因。该研究为快速发掘利用云南野生稻中的抗虫基因奠定了理论基础。