Combining bacterial blight resistance and Basmati quality characteristics by phenotypic and molecular marker-assisted selection in rice

Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice in tropical Asia. Since all the Basmati varieties are highly susceptible and the disease is prevalent in the entire Basmati growing region of India, BB is a severe constraint in Basmati rice production. The present study was undertaken with the objective of combining the important Basmati quality traits with resistance to BB by a combination of phenotypic and molecular marker-assisted selection (MAS). Screening of 13 near-isogenic lines of rice against four isolates of the pathogen from Basmati growing regions identified the Xa4, xa8, xa13 and Xa21 effective against all the isolates tested. Two or more of these genes in combination imparted enhanced resistance as expressed by reduced average lesion length in comparison to individual genes. The two-gene pyramid line IRBB55 carrying xa13 and Xa21 was found equally effective as three/four gene pyramid lines. The two BB resistance genes present in IRBB55 were combined with the Basmati quality traits of Pusa Basmati-1 (PB-1), the most popular high yielding Basmati rice variety used as recurrent parent. Phenotypic selection for disease resistance, agronomic and Basmati quality characteristics and marker-assisted selection for the two resistance genes were carried out in BC₁F₁, BC₁F₂ and BC₁F₃ generations. Background analysis using 252 polymorphic amplified fragment length polymorphism (AFLP) markers detected 80.4 to 86.7% recurrent parent alleles in BC₁F₃ selections. Recombinants having enhanced resistance to BB, Basmati quality and desirable agronomic traits were identified, which can either be directly developed into commercial varieties or used as immediate donors of BB resistance in Basmati breeding programs.     

Pyramiding Xa23 and Rxo1 for resistance to two bacterial diseases into an elite indica rice variety using molecular approaches

Rice bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae and bacterial leaf streak (BLS) caused by X. oryzae pv. oryzicola (Xoc) are two important diseases of rice that often outbreak simultaneously and constrain rice production in much of Asia and parts of Africa. Developing resistant cultivars has been the most effective approach to control BB, however, most single resistance genes have limited value in breeding programs because of their narrow-spectrum of resistance to the races of the pathogen. By contrast, there is little progress in breeding varieties resistant to Xoc since BLS resistance in rice was a quantitative trait and so far only a few quantitative resistance loci have been identified. We reported here the development of a high yield elite line, Lu-You-Zhan highly resistant to both BB and BLS by pyramiding Xa23 with a wide-spectrum resistance to BB derived from wild rice and a non-host maize resistance gene, Rxo1, using both marker assisted selection (MAS) and genetic engineering. Our study has provided strong evidence that non-host R genes could be a valuable source of resistance in combating those plant diseases where no single R gene controlling high level of resistance exists and demonstrated that MAS combined with transgenic technologies are an effective strategy to achieve high level of resistance against multiple plant diseases. Y-L Zhou and J-L Xu contributed equally to this work.     

Pyramiding three bacterial blight resistance genes (xa5, xa13 and Xa21) using marker-assisted selection into indica rice cultivar PR106

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in several countries. Three BB resistance genes, xa5, xa13 and Xa21, were pyramided into cv. PR106, which is widely grown in Punjab, India, using marker-assisted selection. Lines of PR106 with pyramided genes were evaluated after inoculation with 17 isolates of the pathogen from the Punjab and six races of Xoo from the Philippines. Genes in combinations were found to provide high levels of resistance to the predominant Xoo isolates from the Punjab and six races from the Philippines. Lines of PR106 with two and three BB resistance genes were also evaluated under natural conditions at 31 sites in commercial fields. The combination of genes provided a wider spectrum of resistance to the pathogen population prevalent in the region; Xa21 was the most effective, followed by xa5. Resistance gene xa13 was the least effective against Xoo. Only 1 of the BB isolates, PX04, was virulent on the line carrying Xa21 but avirulent on the lines having xa5 and xa13 genes in combination with Xa21. Received: 26 May 2000 / Accepted: 16 August 2000     

Induction of bacterial blight (Xanthomonas oryzae pv. oryzae) resistance in rice by treatment with acibenzolar-S-methyl

The role of the plant defence activator, acibenzolar‐S‐methyl (ASM), in inducing resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) was studied. Application of ASM induced resistance in rice to infection by Xoo. When the pathogen was clip‐inoculated to the rice plants, it caused bacterial leaf blight symptoms in the untreated control. However, in the rice plants pretreated with ASM, infection was significantly reduced. Induced systemic resistance was found to persist for up to 3 days in the pretreated rice plants. Increased phenolic content and accumulation of pathogenesis‐related (PR) proteins, viz. chitinase, β‐1,3‐glucanase and thaumatin‐like protein (TLP; PR 5) were observed in rice plants pretreated with ASM followed by inoculation with Xoo. Immunoblot analysis using rice TLP and tobacco chitinase antiserum revealed rapid induction and over‐expression of 25 and 35 kDa TLP and chitinase, respectively, in rice in response to pretreatment with ASM followed by Xoo inoculation. Based on these experiments, it is evident that induction of disease resistance in rice was accelerated following treatment with ASM.     

农杆菌介导将白叶枯病抗性基因Xa21导入水稻获得转基因植株

利用农杆菌介导的高效遗传转化系统,将白叶枯病抗性基因Xa21转入黄淮稻区主栽品种豫粳6号的胚性愈伤组织,获得转基因植株,GUS染色和PCR分析证明Xa21基因已整合到水稻基因组中,其自交T1代植株经GUS染色和白叶枯病接种鉴定呈现3：1分离,研究为培育抗白叶枯病水稻品种奠定了基础。     

Genetic enhancement for semi-dwarf and bacterial blight resistance with enhanced grain quality characteristics in traditional Basmati rice through marker-assisted selection

Ranbir Basmati is one of the traditional Basmati varieties of India and of the most popular traditional Basmati variety grown in Jammu's region (State of Jammu & Kashmir). It is a tall and short-duration variety with strong aroma and excellent cooking quality. However, it is susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv oryzae (Xoo) and prone to lodging. In this study, semi-dwarf (sd1) and BB resistance genes (Xa21 and xa13) were introgressed into Ranbir Basmati using marker-assisted backcross breeding (MABB) scheme. A high-yielding PAU148 carrying Xa21, xa13 and sd1 genes was used as a donor parent. On each generation target, genes were selected, while polymorphic SSR markers were used to select plants having maximum recovery of the recurrent genome. The maximum genome recovery of Ranbir Basmati in BC₂F₂ was 86.9% in introgressed line SBTIL121. The genotypes carrying resistant genes exhibited very high levels of tolerance against BB disease along with good Basmati rice grain quality traits. The agronomic traits of introgressed lines evaluated in the field and the laboratory showed that most of the agro-morphological traits were similar or superior to Ranbir Basmati. The identified lines can be further evaluated and released as Improved Ranbir Basmati variety.     

Marker assisted selection of bacterial blight resistance genes in rice

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae is one of the most important diseases affecting rice production in Asia. We were interested in surveying rice genotypes that are popularly used in the Indian breeding program for conferring resistance to bacterial blight, using 11 STMS and 6 STS markers. The basis of selection of these DNA markers was their close linkage to xa5, xa13, and Xa21 genes and their positions on the rice genetic map relative to bacterial blight resistance genes. Eight lines were found to contain the xa5 gene while two lines contained Xa21 gene and none of the lines contained the xa13 gene with the exception of its near-isogenic line. Using the polymorphic markers obtained in the initial survey, marker-assisted selection was performed in the F3 population of a cross between IR-64 and IET-14444 to detect lines containing multiple resistance genes. Of the 59 progeny lines analyzed, eight lines contained both the resistance genes, xa5 and Xa4.     

Integrating marker-assisted background analysis with foreground selection for pyramiding bacterial blight resistance genes into Basmati rice

Bacterial leaf blight (BB), caused by the bacterium Xanthomonas oryzae pv. Oryzae (Xoo), is the major constraint amongst rice diseases in India. CSR-30 is a very popular high-yielding, salt-tolerant Basmati variety widely grown in Haryana, India, but highly susceptible to BB. In the present study, we have successfully introgressed three BB resistance genes (Xa21, xa13 and xa5) from BB-resistant donor variety IRBB-60 into the BB-susceptible Basmati variety CSR-30 through marker-assisted selection (MAS) exercised with stringent phenotypic selection without compromising the Basmati traits. Background analysis using 131 polymorphic SSR markers revealed that recurrent parent genome (RPG) recovery ranged up to 97.1% among 15 BC₃F₁ three-gene-pyramided genotypes. Based on agronomic evaluation, BB reaction, aroma, percentage recovery of RPG, and grain quality evaluation, four genotypes, viz., IC-R28, IC-R68, IC-R32, and IC-R42, were found promising and advanced to BC₃F₂ generation.     

Breeding bacterial blight-resistant hybrid rice with the cloned bacterial blight resistance gene Xa21

The cloned bacterial blight (BB) resistance gene Xa21 was transferred into Minghui63, a widely used restorer line of indica hybrid rice in China, through an Agrobacterium-mediated system. Molecular and resistance analyses revealed that the Xa21 gene was integrated in the genomes of transgenic plants and their progeny inherited resistance stably. For the purpose of hybrid breeding, Xa21 transgenic homozygous restorer lines were selected through `within-lane' dosage comparison of hybridization signal in combination with PCR and resistance analyses. The selected transgenic restorer lines were then crossed with a commonly used sterile line, Zhenshan97A, to produce Xa21 transgenic hybrid rice, Shanyou63-Xa21. The hybrid rice plants with Xa21 displayed high broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo) races and maintained elite agronomic characters of Shanyou63. The propagation of this BB-resistant hybrid variety with Xa21 will benefit rice production.     

Cocculus hirsutus extract inhibits the Xanthomonas oryzae pv. oryzae,the bacterial leaf blight pathogen in rice

Plant-derived natural bactericides and their possible applications in agriculture to control plant bacterial diseases has intensified as this approach has enormous potential to inspire and influence modern agro-chemical research. Naturally occurring and biologically active plant products such as essential oils and organic extracts could be a source of alternative classes of natural biopesticides to serve as templates for new and more effective compounds in controlling plant pathogenic micro-organisms. In the present study, the efficacy of six plants extracts from different solvent system were tested for their antibacterial activity aganist Xanthomonas oryzae pv. oryzae both in vitro and in vivo. Among these extracts, Cocculus hirsutus leaf chloroform extract exhibits significant antibacterial activity against X. oryzae pv. oryzae. Data obtained from the experiments such as minimum inhibitory concentration, effect of C. hirsutus leaf chloroform extract on the incidence of X. oryzae pv. oryzae, phytotoxicity test and effect of C. hirsutus leaf chloroform extract on seed germination and seedling vigour, along with the in vivo experiments under greenhouse conditions showed significant improvement over controls. Thus, the present study demonstrated that the C. hirsutus leaf chloroform extract posses antibacterial activity against bacterial leaf blight pathogen of rice.     

Introduction of a rice blight resistance gene,Xa21, into five Chinese rice varieties through anAgrobacterium-mediated system

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3 : 1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transfor-mants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.     

水稻对白叶枯病和细菌性条斑病的抗性遗传研究进展

水稻白叶枯病和水稻细菌性条斑病是由稻黄单胞细菌(Xanthomonas oryzae)不同致病变种引起的两种最重要的水稻细菌性病害。发掘和利用抗性基因,培育抗病品种是防治这两种病害的最有效手段之一。本文分别综述了这两种高度相关的病害的抗性遗传研究进展,包括已发掘和利用的主效抗性基因特点及目前国内外对这两种病害的抗性QTL定位研究进展,为水稻抗白叶枯病和细菌性条斑病育种研究提供有用信息。     

Introduction of a rice blight resistance gene, Xa21, into five Chinese rice varieties through an Agrobacterium-mediated system

A cloned gene, Xa21 was transferred into five widely-used Chinese rice varieties through an Agrobacterium-mediated system, and over 110 independent transgenic lines were obtained. PCR and Southern analysis of transgenic plants revealed the integration of the whole Xa21 gene into the host genomes. The integrated Xa21 gene was stably inherited, and segregated in a 3∶1 ratio in the selfed T1 generation when one copy of the gene was integrated in the transformants. Inoculation tests displayed that transgenic T0 plants and Xa21 PCR-positive T1 plants were highly resistant to bacterial blight disease. The selected Xa21 homozygous resistant transgenic lines with desirable qualities may be propagated as new varieties or utilized in hybrid rice breeding.     

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight. S. Maruthasalam and K. Kalpana have contributed to this article equally.     

云南30个水稻品种对水稻白叶枯病的抗性评价

【目的】水稻白叶枯病是一种严重危害水稻的细菌性病害,培育抗性品种是治理该病害的重要途径。因此,明确云南水稻材料对检疫性病害水稻白叶枯病的抗性,可以为该病害的防治与监测、水稻栽培的合理布局和良好抗性资源的获取提供依据。【方法】采用剪叶接种法测定云南稻区30个品种对7个不同致病型白叶枯病菌的抗性。【结果】在供试的30个云南水稻品种中,2个品种(玉粳16和JS42糯稻)对7个不同致病型菌株均表现为抗性;15个品种对7个致病型菌株均表现感病;对HEN11、SCYC-6、YN7、YN11、FUJ、YN241和PX099等7个致病型菌株表现抗性的水稻品种分别占26.67%、16.67%、23.33%、13.33%、6.67%、10.00%和20.00%。此外,区试材料的抗性比例高于主栽品种,地方稻未发现抗性品种。【结论】现在生产上的大部分水稻品种对优势致病型病原菌入侵的抵抗能力降低甚至丧失。针对云南地区的优势致病小种FUJ筛选得到2个抗性品种:玉粳16和JS42糯稻。     

Pathogenesis-related proteins in somatic hybrid rice induced by bacterial blight

Rice bacterial blight, caused by Xanthomonasoryzae pv. Oryzae (Xoo), is one of the most serious rice diseases worldwide. The bacterial blight resistance trait from Oryza meyeriana, a wild rice species, was introduced into an elite japonica rice cultivar using asymmetric somatic hybridization. This study was carried out with the intention of understanding the molecular mechanism of incompatible interaction between Xoo and the stable somatic hybrids by using proteomic analyses. Proteins were extracted from leaves at 24, 48, and 72 h after Xoo inoculation and separated by 2-DE. A total of 77 protein spots changed their intensities significantly (p<0.05) by more than 1.5-fold at least at one time point. Sixty-four protein spots were successfully identified by MS analysis. Among them, 51 were known to be involved in photosynthesis. Up-regulation of Rubisco large subunit (RcbL) small fragments and down-regulation of RcbL big fragments indicated that intact RcbL and RcbL big fragments degraded following Xoo attack, which was further confirmed by Western blot analysis. The differential expression of proteins related to signal transduction, antioxidant defense, photosynthesis, metabolism, and protein turnover during the Xoo infection, suggests the existence of a complex regulatory network in the somatic hybrid rice that increases resistance toward Xoo infection and damage.     

Suppression of bacterial infection in rice by treatment with a sulfated peptide

The rice XA21 receptor kinase confers robust resistance to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). A tyrosine‐sulfated peptide from Xoo, called RaxX, triggers XA21‐mediated immune responses, including the production of ethylene and reactive oxygen species and the induction of defence gene expression. It has not been tested previously whether these responses confer effective resistance to Xoo. Here, we describe a newly established post‐inoculation treatment assay that facilitates investigations into the effect of the sulfated RaxX peptide in planta. In this assay, rice plants were inoculated with a virulent strain of Xoo and then treated with the RaxX peptide 2 days after inoculation. We found that post‐inoculation treatment of XA21 plants with the sulfated RaxX peptide suppresses the development of Xoo infection in XA21 rice plants. The treated plants display restricted lesion development and reduced bacterial growth. Our findings demonstrate that exogenous application of sulfated RaxX activates XA21‐mediated immunity in planta, and provides a potential strategy for the control of bacterial disease in the field.     

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

Induction of systemic resistance in rice to bacterial blight by 1,2,3-benzothiadiazole 7-carbothioic acid-S-methyl ester (BTH) treatments

Use of BTH to evaluate the disease severity and induction of systemic resistance in rice to bacterial blight caused by Xanthomonas oryzae pv. oryzae is investigated. A new batch of 25 isolates of Xanthomonas oryzae pv. oryzae was obtained from infected rice lead tissues collected from Pattambi, Kerala, south India. Their identification was confirmed by the plant inoculation test on to IR24 rice plants which produced characteristic bacterial blight lesions. Among the 25 of X.o. pv. oryzae, four of the isolates were also virulent to IRBB21 rice plants (a near isogenic line of IR24) which carry the Xa-21 gene for BB resistance. The results confirm that there are pathogen strains in India which can overcome Xa-21. Development of BB lesions developed in IR24 (BB susceptible) plants after they were treated with BTH applications either as seed treatment or as foliar spray at 0.1, 0.5, 0.1 and 2.0 mM concentrations showed that even at 2.0 mM concentrations, IR24 plants were still susceptible to the pathogen. There was very little or marginal effect of BTH on the induction of resistance to BB in IR24 rice plants. When the same concentrations of BTH were applied to IRBB21 (Xa-21) rice plants, they showed pronounced triggering of systemic resistance to BB pathogen even at 0.1 mM concentration of BTH applied either as seed treatment or as foliar spry. Disease severity index was reduced to 5 (against a score of 9 in untreated) and there was 85–86% reduction in BB incidence in plants that received 0.1 mM BTH. These results provide evidence that BTH-induced systemic resistance complements the R-gene resistance in IRBB21 plants but not in IR24 rice plants.