Molecular mapping of rice blast resistance gene <Emphasis Type="Italic">Pi-1</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) in the elite <Emphasis Type="Italic">indica</Emphasis> variety Samba mahsuri

Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation ratio of 3:1 for resistant to susceptible F₂ plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001.     

施硅增强水稻对纹枯病的抗性

采用水培的方法,从细胞学和生理生化方面研究了硅增强水稻对纹枯病的抗性作用。结果表明：加硅处理的水稻叶片硅化细胞和叶片表面的硅元素含量均显著高于缺硅处理（对照）：接种纹枯病菌后,加硅处理的MDA含量总体上低于缺硅处理,峰值尤为显著;加硅处理的SOD活性始终高于缺硅处理,接种后第4天加硅处理SOD活性较低时,其POD活性较高,而缺硅处理的POD活性较低,表明硅增强了SOD和POD之间的协调性;接种后硅对CAT和PAL活性没有产生明显影响,但降低了PP0活性;加硅能显著降低水稻植株的纹枯病病情指数。     

Silicon enhances photochemical efficiency and adjusts mineral nutrient absorption in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> infected rice plants

Silicon (Si) has been verified to play an important role in enhancing plant resistance against pathogens, but the exact mechanisms remain unclear. Two near-isogenic lines of rice (Oryza sativa L.), CO39 (blast susceptible), and C101LAC (Pi-1) (blast resistant), were hydroponically grown to study the effects of exogenous silicon application on the changes of disease incidence, mineral nutrient concentrations, chlorophyll content, and photochemical efficiency in Magnaporthe oryzae infected rice plants. Si amendment in nutrient solution at a concentration of 2.0 mM significantly reduced the disease index of rice plants of CO39 and C101LAC (Pi-1). Silicon application alone had no effects on mineral nutrient contents, chlorophyll content, maximum/potential quantum efficiency (F _v/F _m), and the maximum primary yield (F _v/F ₀) of photochemistry of PS II in healthy rice leaves. M. oryzae inoculation significantly increased the content of K, Na, Ca, Mg, Fe, and reduced the value of F _v/F ₀ and F _v/F _m in rice leaves. However, Si treatment suppressed M. oryzae induced increase of mineral nutrient contents, and significantly increased F _v/F ₀ and F _v/F _m value compared with Si-deficient infected plants. These results suggest that silicon-enhanced resistance to rice blast is associated with an enhancement of photochemical efficiency and adjustment of mineral nutrient absorption in M. oryzae-infected rice plants.     

Physiological Responses of Rice Plants Supplied with Silicon to Monographella albescens Infection

This study investigated the effect of silicon (Si) on the resistance of rice plants of the cv. ‘Primavera’ cultivar that were grown in a nutrient solution with 0 (?Si) or 2 mm (+Si) Si to leaf scald, which is caused by Monographella albescens. The leaf Si concentration increased in the +Si plants (4.8 decag/kg) compared to the ?Si plants (0.9 decag/kg), contributing to a reduced expansion of the leaf scald lesions. The extent of the cellular damage that was caused by the oxidative burst in response to the infection by M. albescens was reduced in the +Si plants, as evidenced by the reduced concentration of malondialdehyde. Higher concentrations of total soluble phenolics and lignin‐thioglycolic acid derivatives and greater activities of peroxidases (POX), polyphenoloxidases (PPO), phenylalanine ammonia‐lyases (PAL) and lipoxygenases (LOX) in the leaves of the +Si plants also contributed to the increased rice resistance to leaf scald. In contrast, the activities of chitinases and β‐1,3‐glucanases were higher in the leaves of the ?Si plants probably due to the unlimited M. albescens growth in the leaf tissues, as indicated by the larger lesions. The results of this study highlight the potential of Si in decreasing the expansion of the leaf scald lesions concomitantly with the potentiation of phenolic and lignin production, and the greater activities of POX, PPO, PAL and LOX rather than simply acting only as a physical barrier to avoid M. albescens penetration.     

Azoxystrobin induces lignification-related enzymes and phenolics in rice (Oryza sativa L.) against blast pathogen (Pyricularia grisea)

Abstract

This study was conducted to analyse the induction of lignification-related enzymes and phenolic content in rice to blast disease caused by Pyricularia grisea using azoxystrobin. The severity of rice blast was reduced (70% over control) through treatment by azoxystrobin. This reduction in disease severity was mainly associated with induction of host defense mechanisms by azoxystrobin. Increased production of secondary metabolite – phenolic and lignification – related enzymes, namely, peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were observed in rice plants treated with azoxystrobin.     

Identification and fine mapping of <Emphasis Type="Italic">Pi33</Emphasis>, the rice resistance gene corresponding to the Magnaporthe grisea avirulence gene <Emphasis Type="Italic">ACE1</Emphasis>

Rice blast disease is a major constraint for rice breeding. Nevertheless, the genetic basis of resistance remains poorly understood for most rice varieties, and new resistance genes remain to be identified. We identified the resistance gene corresponding to the cloned avirulence gene ACE1 using pairs of isogenic strains of Magnaporthe grisea differing only by their ACE1 allele. This resistance gene was mapped on the short arm of rice chromosome 8 using progenies from the crosses IR64 (resistant) × Azucena (susceptible) and Azucena × Bala (resistant). The isogenic strains also permitted the detection of this resistance gene in several rice varieties, including the differential isogenic line C101LAC. Allelism tests permitted us to distinguish this gene from two other resistance genes [Pi11 and Pi-29(t)] that are present on the short arm of chromosome 8. Segregation analysis in F₂ populations was in agreement with the existence of a single dominant gene, designated as Pi33. Finally, Pi33 was finely mapped between two molecular markers of the rice genetic map that are separated by a distance of 1.6 cM. Detection of Pi33 in different semi-dwarf indica varieties indicated that this gene could originate from either one or a few varieties.Communicated by D.J. Mackill     

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.     

稻瘟病菌T-DNA插入方法优化及其突变体分析

优化了农杆菌介导转化稻瘟病菌获得T-DNA插入突变的条件,包括选择转化子的潮霉素B用量,抑制农杆菌的抗生素头孢噻肟钠和羧苄青霉素的配比,不同转化阶段培养基的选择等。转化1×10⁶个孢子平均可获得约500个左右的转化子,PCR和TAILPCR检测表明约85%转化子中含T-DNA插入。对1520个突变体进行形态变异观察,发现菌落颜色突变的有15个;随机取58个突变体进行比较,发现产孢量减少的4个,孢子萌发率降低的8个,附着胞形成率降低的9个;还获得对水稻品种C101LAC（Pi-1）和751127（Pi-9）致病的突变体,为进一步克隆相应的无毒基因奠定了基础。     

湖南桃江病圃稻瘟病菌的无毒基因及水稻抗瘟单基因联合抗性分析

【目的】鉴定湖南省桃江病圃稻瘟病菌无毒基因型,为合理搭配种植湖南省水稻抗瘟品种和抗病育种提供依据。【方法】在湖南桃江病圃采集水稻品种"丽江新团黑谷"(LTH)稻瘟菌病样,用单孢分离法分离稻瘟病菌单孢并纯化获得单孢菌株,用针刺离体法将菌株接种到以"LTH"为轮回亲本培育而成的24个含单抗瘟基因的水稻5叶期第5叶片上,对供试菌株进行无毒基因鉴定,并应用联合致病性系数和联合抗病性系数分析抗瘟基因组合间的互作。【结果】供试92个稻瘟病单孢菌株含有全部的24个无毒基因,对24个已知含单抗瘟基因的水稻材料表现出不同程度的毒力水平,含水稻抗瘟基因Pi-20对供试菌株抗菌频率最高,达54.35%;通过联合致病性系数和联合抗病性系数分析抗瘟基因组合间的互作,结果表明最佳搭配组合为Pi-20×Pi-k~s(RAC=0.28,PAC=0.23)。【结论】湖南省桃江病圃稻瘟病菌致病力较强,24个抗瘟基因多已感病化,含抗性基因Pi-20与Pi-k、Pi-k~s、Pi-3组合的水稻品种目前可在湖南省推广利用,但需研究引进新的抗瘟基因。     

核黄素和接种番茄黄化曲叶病毒对番茄叶片防御酶活性的影响

以番茄品种‘1479’为材料,研究了喷施核黄素(Riboflavin)和接种番茄黄化曲叶病毒(TYLCV)对幼苗叶片中过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)3种防御酶活性的影响。结果显示:(1)核黄素能显著降低番茄植株的番茄黄化曲叶病毒病的病情指数,并以2 mmol/L时诱导效果最佳,诱抗效果最高可达41.91%。(2)2mmol/L核黄素处理后96h内,番茄叶片的POD、PPO和PAL活性显著高于对照。(3)接种TYLCV后,核黄素处理和接种TYLCV处理均可诱导番茄叶片中防御酶活性显著增强。研究表明,核黄素处理可诱导POD、PPO和PAL活性的系统增强与番茄对TYLCV的诱导抗性密切相关。     

苗龄、光照强度和施氮量对抗褐飞虱水稻品种主要防御酶活性的影响

为探明苗龄、光照强度和施氮量对水稻主要防御酶活性的影响及其与抗褐飞虱的关系,分别就苗龄、光照强度和施氮量等因子对不同抗褐飞虱水稻品种苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)和过氧化物酶(POD)活性的影响进行研究。结果表明:IR56、Ptb33和570011对褐飞虱生物型II的抗性与PAL和PPO活性密切相关,RHT对II型的抗性与PAL、PPO和POD活性密切相关,RP1976-18-6-4-2对II型的抗性与PAL活性密切相关,570011对II型的抗性与PAL和PPO活性密切相关;IR56对褐飞虱孟加拉型的抗性与PAL、PPO和POD活性密切相关,Rathu Heenati(RHT)和RP1976-18-6-4-2对孟加拉型的抗性与与PPO密切相关,570011对孟加拉型的抗性与POD活性密切相关。苗龄、光照强度和施氮量的单独作用或交互作用对不同水稻中防御酶活性有明显影响,但品种不同,其影响程度也不同。苗龄、光照强度和施氮量单独或是交互作用显著影响IR56中PAL和PPO、RHT和RP1976-18-6-4-2中PPO、Ptb33中POD及570011中PAL和POD的活性,苗龄显著影响IR56和RP1976-18-6-4-2中POD的活性,光照强度和施氮量单独或交互作用显著影响RHT和RP1976-18-6-4-2中PAL的活性,施氮量显著影响RHT中POD的活性,光照强度显著影响Ptb33中PAL的活性;苗龄和光照强度单独或交互作用显著影响570011中PPO的活性。     

Function of silica bodies in the epidermal system of rice (Oryza sativa L.): testing the window hypothesis

Silicon has been considered to be important for normal growth and development of the rice plant (Oryza sativa L.). To investigate the physiological function of deposited silica in rice leaves, the hypothesis that silica bodies in the leaf epidermal system might act as a 'window' to facilitate the transmission of light to photosynthetic mesophyll tissue was tested. The silica content of leaves increased with supplied silicon and was closely correlated with the number of silica bodies per unit leaf area in the epidermal system. There was a significant difference in silica deposition and formation of silica bodies between Si-treated and non-treated leaves; silicon was polymerized inside the silica cells and bulliform cells of the epidermis, in Si-treated leaves. Although the 'windows' were only formed in leaves with applied silicon, optical properties of leaf transmittance, reflectance and absorptance spectra in Si-treated and non-treated leaves were almost equal. Furthermore, light energy use efficiency and quantum yield of Si-treated leaves were less than in leaves not containing silica bodies. Thus, silica bodies, at least based on the data, do not function as windows in rice leaves.     

不同番木瓜品种植株感染环斑花叶病毒后PAL、PPO、POD活性的变化

对不同品种番木瓜接种环斑花叶病毒后,测定植株苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、过氧化物酶(POD)的活性变化,比较各品种的抗病性。结果表明,未接种的5个品种间PAL、PPO、POD活性差异较小,其酶活性水平次序为马来10号> 蜜红3号> 马来6号> 马来2号> 台农2号。接种后,5个品种的PAL、PPO、POD活性明显高于各对照水平,其中马来10号变化最突出,台农2号变化最缓慢。     

Identification of a 118-kb DNA fragment containing the locus of blast resistance gene Pi-2(t) in rice

Rice blast disease, caused by the fungal pathogen Pyricularia grisea Sacc., is one of the most devastating crop diseases worldwide. Previous studies have shown that the dominant blast resistance gene Pi-2(t) confers resistance to a broad spectrum of pathogenic strains. Using a population of 292 recombinant inbred lines combined with bioinformatic analysis, we mapped Pi-2(t) between the SSR (simple-sequence repeat) marker SSR140 and the RFLP (restriction fragment length polymorphism) marker JSH12, 0.9 cM from both SSR140 and JSH12. A physical map consisting of six overlapping BAC (bacterial artificial chromosome) clones was anchored to the region containing the Pi-2(t) locus. By analyzing recombination events in this region, the Pi-2(t) locus was localized to a DNA fragment of 118 kb in length. The detailed genetic and physical maps of the Pi-2(t) locus will facilitate both molecular isolation of the gene and marker-assisted transfer of the gene in breeding programs.     

绿盲蝽取食与机械损伤对棉花叶片内防御性酶活性的影响

为探明绿盲蝽Apolygus lucorum (Mayer-Dür)取食和机械损伤对不同抗性棉花叶片内主要防御酶活性的影响以及防御酶与棉花抗绿盲蝽性的关系,以棉花3个不同抗性品系为材料,室内条件下测定绿盲蝽取食和机械损伤处理后棉叶中苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)和多酚氧化酶(PPO)的活性.结果表明:对...     

不同基因型梨木质素代谢对石细胞含量和果实口感的影响

以五种基因型梨果实为材料,对石细胞团的大小、分布进行解剖学观察,并测定石细胞、木质素含量和木质素相关合成酶PAL、POD、PPO活性,探讨不同基因型梨果实木质素代谢对石细胞含量及口感的影响.结果表明,不同基因型梨木质素含量高时,石细胞含量也高,石细胞团相对较大,分布较密集,口感差.各基因型的梨木质素、石细胞含量和大小为...     

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.     

甘蓝幼苗受桃蚜危害后叶片中部分酶活性的变化

对桃蚜（Myzus persicae Sulzer）危害后甘蓝（Brassica oleracea L.）幼苗叶中多酚氧化酶（PPO）、苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）活性及同工酶进行了研究。结果表明,随桃蚜危害时间延长,处理叶片PPO、PAL和POD的活性与对照相比均表现出升高的趋势,方差分析表明与对照之间差异显著。同工酶电泳结果表明,POD同工酶的部分谱带随危害时间而发生变化,但EST同工酶在接虫前后则无明显的变化。     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.