Brown planthopper, Nilaparvata lugens, resistance in rice cultivar IR64: mechanism and role in successful N. lugens management in Central Luzon, Philippines

We investigated the mechanism of brown planthopper, Nilaparvata lugens (Stål), resistance in the popular rice cultivar IR64, and the current level of resistance of IR64 to N. lugens in a large rice growing area with low brown planthopper populations (Central Luzon, Philippines). In greenhouse experiments with N. lugens populations collected from Central Luzon, IR64 showed slight to moderate levels of antibiosis, antixenosis, and tolerance relative to the cultivars IR22 and Azucena, which contain no major genes for N. lugens resistance. IR64 was also more resistant than IR26 in most experiments, despite the fact that both varieties have the same major gene for N. lugens resistance, Bph1. This confirms that IR64 contains one or more additional, apparently minor, genes for brown planthopper resistance. Our findings also demonstrate that, in an area with low insecticide use, it is not necessary to have high levels of N. lugens resistance, even in a rice cultivar grown by the majority of farmers, to maintain low and stable N. lugens populations.     

Aspects of brown planthopper adaptation to resistant rice varieties with the Bph3 gene

Despite over 30 years of deployment, varieties with the Bph3 gene for resistance to the brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), are still effective in much of the Philippines. In the present study, we determined the effects of adaptation to one resistant variety, IR62 – assumed to possess the Bph3 gene – on (1) resistance against a series of varieties with similar biotypical responses (presumed to contain the same major resistance genes), and (2) a differential variety with the bph4 gene that occurs at the same chromosome position as Bph3. We also examined the effects of high soil nitrogen on the effectiveness of Bph3. Feeding, planthopper biomass, and development times were reduced in a wild BPH population when reared on IR62 compared with the susceptible standard variety TN1. However, nitrogen application increased the susceptibility of IR62. After 13 generations on IR62, BPH had adapted to the plant’s resistance. Virulence of the adapted BPH against the variety ‘Rathu Heenati’ supports the idea that Bph3 is present in IR62. Across similar IR varieties (IR60, IR66, IR68, IR70, IR72, and IR74), feeding, planthopper biomass, and development rates were generally higher for IR62‐adapted than for non‐adapted BPH; however, contrary to expectations, many of these varieties were already susceptible to wild BPH. Fitness was also higher for IR62‐adapted BPH on the variety ‘Babawee’ indicating a close relation between Bph3 and bph4. The results indicate that the conventional understanding of the genetics behind resistance in IR varieties needs to be readdressed to develop and improve deployment strategies for resistance management.     

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.     

Detection and analysis of QTLs for resistance to the brown planthopper, Nilaparvata lugens, in a doubled-haploid rice population

 We used a mapping population of 131 doubled-haploid lines, produced from a cross between an improved indica rice variety (IR64) and a traditional japonica variety (Azucena), to detect quantitative trait loci (QTLs) for resistance to the brown planthopper (BPH), Nilaparvata lugens. We evaluated the parents and mapping population with six tests that measure varying combinations of the three basic mechanisms of insect host plant resistance, i.e., antixenosis, antibiosis, and tolerance. To factor-out the effect of the major resistance gene Bph1 from IR64, the screening was done with two BPH populations from Luzon Island, The Philippines, that are almost completely adapted to this gene. A total of seven QTLs associated with resistance were identified, located on 6 of the 12 rice chromosomes. Individual QTLs accounted for between 5.1 and 16.6% of the phenotypic variance. Two QTLs were predominantly associated with a single resistance mechanism: one with antixenosis and one with tolerance. Most of the QTLs were derived from IR64, which has been shown to have a relatively durable level of moderate resistance under field conditions. The results of this study should be useful in transferring this resistance to additional rice varieties. Received: 10 May 1998 / Accepted: 4 June 1998     

不同地理种群褐飞虱的抗逆性

研究了不同地理种群褐飞虱对水稻抗性品种、若虫高密度、高温、饥饿和杀虫剂等逆境条件的抗性。结果表明,广西和云南种群褐飞虱对抗虫品种的适应性比浙江种群强而广,浙江种群只能适应抗虫品种ＩＲ２６而广西和云南种群则还能适应抗性更高的ＡＳＤ７和Ｒａｔｈｕ Ｈｅｅｎａｔｉ。广西和云南种群较能适应高密度环境,而浙江种群的适应性较差,其雌虫短翅率与若虫密度呈显著负相关。虽然广西和云南种群对高温的忍受性比浙江种群的强     

Influence of pymetrozine on feeding behaviors of three rice planthoppers and a rice leafhopper using electrical penetration graphs

Pymetrozine reportedly inhibits feeding of plant sap-sucking insects, such as aphids and brown planthopper (Nilaparvata lugens (St?l)). By using electrical penetration graph (EPG), this study was conducted to investigate any differential effect of pymetrozine on the feeding behaviors of four major rice sap-sucking insect species, 1) N. lugens, 2) white-backed planthopper (Sogatella furcifera (Horváth)), 3) small brown planthopper (Laodelphax striatellus (Fallen)), and 4) green rice leafhopper (Nephotettix cincticeps Uhler). On pymetrozine-free TN1 rice plants, white-backed planthopper and small brown planthopper showed a significantly less activity in the phloem phases than brown planthopper or green rice leafhopper while green rice leafhopper engaged in relatively more xylem ingestion than brown planthopper, white-backed planthopper, and small brown planthopper. On the plants treated with 100 mg liter(-1) of pymetrozine, all four insect species showed significant increases, in total duration of nonprobing and significant decreases in the activities in phloem tissue, while all species showed similar feeding behavior during the pathway and xylem phases. This study revealed that, regardless of whether the insects on untreated plants spent more time feeding on phloem than xylem (brown planthopper) or more time on xylem than phloem (green rice leafhopper) or similar times on phloem and xylem (white-backed planthopper and small brown planthopper), their feeding behavior was disturbed by pymetrozine and exhibited similar patterns of sharp decline in activity in the phloem tissue and a significant increase the nonprobing.     

Categories and inheritance of resistance to Nilaparvata lugens (Hemiptera: Delphacidae) in mutants of indica rice 'IR64'

Varietal mutants can be useful for developing durable resistance, understanding categories of resistance, and identifying candidate genes involved in defense responses. We used mutants of rice 'IR64' to isolate new sources of resistance to the planthopper Nilaparvata lugens (St?l) (Hemiptera: Delphacidae). We compared two mutants that showed a gain and loss of resistance to N. lugens, to determine the categories of resistance to this pest. Under choice tests, female planthoppers avoided settling and laid fewer eggs on the resistant mutant 'D518' than on the susceptible mutant D1131, susceptible check 'TN1', and wild-type IR64, indicating that antixenosis was the resistance category. Similarly, under no-choice conditions, planthoppers laid 29% fewer eggs in D518 than in IR64, but they oviposited more in 'D1131' and TN1. Honeydew excretion was greater on D1131 seedlings but slightly lower on D518 than on IR64. Nymphal survival and adult female weight did not differ among rice cultivars. D518 showed higher tolerance of N. lugens infestations than IR64. Genetic analysis of the F1, F2, and F3 populations derived from D518 x IR64 revealed that resistance in D518 is dominant and controlled by a single gene. Despite the variation in resistance to N. lugens, both mutants and IR64 performed similarly in the field. The mutant D518 is a new source of durable resistance to N. lugens, mainly due to enhanced antixenosis to female hoppers for settling and oviposition.     

重庆市秀山县稻飞虱发生特点及其原因分析

重庆市秀山地区稻飞虱的发生主害种类为白背飞虱Sogatellafurcifera和褐飞虱Nilaparvatalugens,白背飞虱的危害重于褐飞虱 ;白背飞虱 1年发生 5～ 6代 ,以 4 ( 3)代为主害代 ,田间发生 2～ 3次若虫高峰 ,以第 2次为主害代峰、虫量最大 ;褐飞虱 1年发生 4～ 5代 ,以 5 ( 4 )代为主害代 ,田间发生 3～ 4次若虫高峰 ,以第 3次为主害代峰、虫量最大 ;( 3)稻飞虱重发年比例高 ,达 85 %以上 ;( 4 )白背飞虱呈逐年加重和主害期提前、褐飞虱呈逐年减轻和主害期推迟的趋势。其主要原因是 :特殊的槽谷地形、迁入虫量大、品种及栽培技术的变化、防治技术的提高和气候条件有利等。     

白背飞虱的迁飞生物学:安徽徽州个例分析

通过水稻田罩笼试验研究了白背飞虱起飞迁出与世代、田间成虫密度的关系以及降水对起飞的影响,采用系统田调查和长翅雌虫卵巢系统解剖分析了各代翅型分化、迁出与水稻生育期的关系以及各世代虫源性质。结果表明:(1)早中稻田白背飞虱起飞迁出以第2、3代为主,后期迁出较少;(2)白背飞虱起飞迁出数量与田间成虫密度有关;(3)降水影响白背飞虱的起飞;(4)白背飞虱各世代以长翅型为主,世代间翅型分化模式不同于褐飞虱,表现为"长—长—长";(5)白背飞虱起飞与水稻生育期关系不大;(6)白背飞虱虫源性质分为基本迁入型、部分迁入滞留与部分迁出型、基本迁出型。     

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.     

Development and use of EST-SSR markers for assessing genetic diversity in the brown planthopper (Nilaparvata lugens Stål)

To assess genetic diversity in populations of the brown planthopper (Nilaparvata lugens St?l) (Homoptera: Delphacidae), we have developed and applied microsatellite, or simple sequence repeat (SSR), markers from expressed sequence tags (ESTs). We found that the brown planthopper clusters of ESTs were rich in SSRs with unique frequencies and distributions of SSR motifs. Three hundred and fifty-one EST-SSR markers were developed and yielded clear bands from samples of four brown planthopper populations. High cross-species transferability of these markers was detected in the closely related planthopper N. muiri. The newly developed EST-SSR markers provided sufficient resolution to distinguish within and among biotypes. Analyses based on SSR data revealed host resistance-based genetic differentiation among different brown planthopper populations; the genetic diversity of populations feeding on susceptible rice varieties was lower than that of populations feeding on resistant rice varieties. This is the first large-scale development of brown planthopper SSR markers, which will be useful for future molecular genetics and genomics studies of this serious agricultural pest.     

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.     

褐飞虱Nl15基因的克隆及功能分析

【目的】植食性刺吸式口器昆虫的唾液蛋白参与调控植物抗虫防御反应,影响其对寄主植物的适应性。本研究旨在通过克隆褐飞虱Nilaparvata lugens重要唾液蛋白基因Nl15,调查其时空表达模式,明确其在褐飞虱致害性中的作用。【方法】基于褐飞虱IR56种群转录组数据,用RT-PCR克隆褐飞虱基因Nl15 cDNA序列,并进行生物信息学分析。利用qPCR检测其在褐飞虱TN1和IR56种群不同发育阶段(卵、1-5龄若虫和雌雄成虫)和雌成虫不同组织(头、胸、腹和足)中的表达模式。通过显微注射dsRNA对褐飞虱TN1和IR56种群的4龄若虫进行Nl15的RNAi,利用qPCR检测Nl15 RNAi后褐飞虱若虫中Nl15的相对表达量以及Nl15 RNAi后褐飞虱若虫取食3 d时水稻植株中防御相关基因(OsLecRK4, OsMPK10, OsWRKY24, OsLox, OsNPR1和OsGns5)的相对表达量,并生物测定Nl15 RNAi后褐飞虱的存活率以及成虫蜜露量和体质量增量。【结果】克隆了褐飞虱Nl15 cDNA序列(GenBank登录号：OK181113),其开放阅读框长1 008 bp;预测编码335个氨基酸,理论等电点为7.54,分子量为38.7 kD,含有23个氨基酸的信号肽序列和一个糖基化修饰位点,不存在跨膜结构域和其他已知的功能域;Nl15与灰飞虱Laodelphax striatellus同源蛋白氨基酸序列一致性为45%。发育表达谱结果表明,Nl15在褐飞虱各个发育阶段均表达,在3-4龄若虫中的表达量最高;组织表达谱结果表明,Nl15在褐飞虱雌成虫头部中的表达量最高,且在IR56种群头部中的表达量高于在TN1种群头部中的。RNAi实验结果表明,与注射dsGFP的对照组相比,注射dsNl15的处理组中Nl15的表达量显著降低了89.5%,褐飞虱的存活率以及成虫蜜露量和体质量增量均显著降低,上述6个水稻防御相关基因的表达量显著上调。【结论】褐飞虱IR56种群中的Nl15参与褐飞虱与水稻的防御与反防御分子互作。本研究为进一步阐述褐飞虱克服抗虫基因的机制及揭示昆虫与植物互作的分子网络提供了思路。     

吡虫啉对褐飞虱DNA甲基化多态性的影响

为了探知基因组甲基化是否参与了昆虫抗药性, 本研究在室内对褐飞虱 Nilaparvata lugens连续9个世代的3龄若虫施用吡虫啉, 用AFLP检测褐飞虱抗性产生过程中DNA甲基化多态性的变化。利用25对AFLP引物共获得120个位点, 其中15个位点呈现甲基化多态性, 共获得78条多态性条带。根据多态性条带在不同世代样本中出现的多少计算多态性条带比例, 其中最高比例出现在G5代（10.26%）, 最低比例出现在G6代（1.28%）。多态性条带在不同世代间比例的变化趋势表明, 褐飞虱对吡虫啉的筛选产生快速应答。在筛选早期（G1, G2和G3）世代间DNA甲基化多态性比例差异相对较小, 变化范围在3.85%～6.41%之间; 在筛选中期（G4, G5和G6）世代间比例差异较大, 变化范围在1.28%～10.26%之间; 在筛选后期（G7, G8和G9）世代间比例差异相对较小, 变化范围在5.13%～7.69%之间。结果说明, 吡虫啉的连续施用能够诱导褐飞虱基因组产生甲基化变异, 初步揭示甲基化在褐飞虱抗药性产生过程中参与了基因组的表达调控。     

小黄家蚁工蚁对褐稻虱的捕食作用

报道了小黄家蚁对褐稻虱的捕食作用．养虫笼及大试管捕食试验结果表明,小黄家蚁对褐稻虱若虫尤其是低龄若虫具有良好的捕食效果,捕食率分别为９３％和３０％,捕食的功能反应为ＨｏｌｉｎｇⅡ型反应,水稻品种抗性与捕食反应之间存在协同作用．     

Fine mapping of the rice Bph1 gene, which confers resistance to the brown planthopper (Nilaparvata lugens stal), and development of STS markers for marker-assisted selection

The brown planthopper (BPH) is a major insect pest in rice, and damages these plants by sucking phloem-sap and transmitting viral diseases. Many BPH resistance genes have been identified in indica varieties and wild rice accessions, but none has yet been cloned. In the present study we report fine mapping of the region containing the Bph1 locus, which enabled us to perform marker-aided selection (MAS). We used 273 F8 recombinant inbred lines (RILs) derived from a cross between Cheongcheongbyeo, an indica type variety harboring Bph1 from Mudgo, and Hwayeongbyeo, a BPH susceptible japonica variety. By random amplification of polymorphic DNA (RAPD) analysis using 656 random 10-mer primers, three RAPD markers (OPH09, OPA10 and OPA15) linked to Bph1 were identified and converted to SCAR (sequence characterized amplified region) markers. These markers were found to be contained in two BAC clones derived from chromosome 12: OPH09 on OSJNBa0011B18, and both OPA10 and OPA15 on OSJNBa0040E10. By sequence analysis of ten additional BAC clones evenly distributed between OSJNBa0011B18 and OSJNBa0040E10, we developed 15 STS markers. Of these, pBPH4 and pBPH14 flanked Bph1 at distances of 0.2 cM and 0.8 cM, respectively. The STS markers pBPH9, pBPH19, pBPH20, and pBPH21 co-segregated with Bph1. These markers were shown to be very useful for marker-assisted selection (MAS) in breeding populations of 32 F6 RILs from a cross between Andabyeo and IR71190, and 32 F5 RILs from a cross between Andabyeo and Suwon452.     

利用SSR定位籼稻品种Kaharamana中抗褐飞虱基因Bph9

褐飞虱是危害水稻生产最重要的害虫之一,利用寄主抗性被认为是防治褐飞虱最经济而有效的方法。斯里兰卡水稻品种Kaharamana对东亚和东南亚的褐飞虱种群均表现抗虫性,利用分子遗传学的方法对其携带的Bph9基因进行了SSR定位。所用的遗传群体为来源于Kaharamana和02428的含有180个单株的F2分离群体,每个F2单株套袋自交获得F2：3家系。利用苗期集团鉴定埘F2：3家系进行抗褐飞虱鉴定,以推测相应F2单株的基因型。连锁分析表明,Bph9位于第12染色体上的两个SSR标记RM463和RM5341之间,分别与之相距6．8cM和9．7cM。该标记有助十将Bph9用于分子标记辅助选择育种研究。     

基因聚合改良杂交稻组合的稻飞虱田间抗性表现

褐飞虱Nilaparvata lugens(Stl)是杂交水稻的重要虫害,培育高抗褐飞虱的杂交水稻新组合被认为是目前防治飞虱最有效、经济的方法。本研究以分子标记辅助选择培育的抗褐飞虱杂交水稻为材料,通过苗期和成株期的全生育期田间抗性鉴定,对基因聚合改良杂交稻组合的褐飞虱抗性进行综合评价,结果表明,基因聚合改良杂交稻组合的苗期抗性介于抗和中抗水平,田间虫量在7次调查中均低于800头/百丛,远低于2000头/百丛的防治标准,而对照扬两优6号、两优培九和当地农家品种两优63在8月29日到9月10日飞虱发生高峰期的百丛虫量分别最高达到5400头、2030头和10700头,高于或远高于2000头/百丛的防治标准。改良组合川香29A/华恢1462的产量在通城比对照扬两优6号增产11.8%。上述结果表明抗褐飞虱改良杂交稻新组合能够有效抑制稻田褐飞虱种群数量,节约人工和农药成本,增加水稻产量。     

褐飞虱为害对不同抗性水稻品种苗期根系几种营养物质和总酚含量的影响

本文以对褐飞虱Nilaparvata lugens(St?l)具不同抗性水平的水稻品种为研究对象,通过在苗期接入不同密度褐飞虱后测定水稻根系可溶性糖、游离氨基酸、可溶性蛋白质和总酚变化情况,以明确水稻抗虫性与根系生理生化物质含量的关系.研究结果表明,根系中的可溶性糖除了苗龄8d不接虫的处理,其余处理均以感虫品种TN1含量最高,显著高于3个抗虫品种IR36、Rathu-Heenati(RHT)、Ptb33.各苗龄不同接虫密度TN1根系游离氨基酸及可溶性蛋白质含量均显著高于3个抗虫品种.各苗龄不同接虫密度TN1根系总酚含量均显著低于3个抗虫品种,以Ptb33总酚含量最高,各处理均显著高于IR36及RHT.结果可为进一步开发利用根系指标优良的抗性水稻品种提供科学依据.