Analysis of Pathogenic Diversity of the Rice Bacterial Blight Pathogen (Xanthomonas oryzae pv. oryzae) in the Andaman Islands and Identification of Effective Resistance Genes

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a major disease of rice in the tropics for which genetic resistance in the host plants is the only effective solution. This study aimed at identification of resistance gene combinations effective against Xoo isolates and fingerprinting of the Xoo isolates of Andaman Islands (India). Here, we report the reaction of 21 rice BB differentials possessing Xa1 to Xa21 genes individually and in different combinations to various isolates of pathogen collected from Andaman Islands. Pathological screening results of 14 isolates revealed that among individual genes tested across 2 years, Xa4, Xa7 and Xa21 conferred resistance reaction across all isolates, whereas among combinations, IRBB 50 (Xa4 + xa5), IRBB 52 (Xa4 + Xa21) and IRBB 60 (Xa4 + xa5 + xa13 + Xa21) conveyed effective resistance against tested isolates. The nature of genetic diversity among four isolates selected on the basis of geographical isolation in the islands was studied through DNA finger printing. The RAPD primers S111, S119, S1117, S1109, S1103, S109 and S105 were found to be better indicators of molecular diversity among isolates than JEL primers. The diversity analysis grouped 14 isolates into three major clusters based on disease reaction wherein isolate no. 8 was found the most divergent as well as highly virulent. The remaining isolates were classified into two distinct groups. The importance of the study in the context of transfer of resistance gene(s) in the local cultivars specifically for tropical island conditions is presented and discussed.     

Pathotypic variation of Xanthomonas oryzae pv. oryzae in Bangladesh

Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo), a destructive disease of rice. Altogether, 96 isolates of Xoo were collected from 19 rice growing districts of Bangladesh in irrigated and rainfed seasons during 2014 to assess pathotypic variation. Pathotypic analyses on a set of 12 Near Isogenic Lines (NILs) of rice containing resistance genes viz. Xa1, Xa2, Xa3, Xa4, Xa5, Xa7, Xa8, Xa10, Xa11, Xa13, Xa14 and Xa21 and two check varieties IR24 and TN1 by leaf clip-inoculation technique. A total of 24 pathotypes were identified based on their virulence patterns on NILs tested. Among these, pathotypes VII, XII, and XIV considered as major, containing maximum number of isolates, (9.38% each) frequently distributed in North to Mid-Eastern districts of Bangladesh. Most virulent pathotype I recorded from Habiganj and Brahmanbaria. This pathotypic variation explained the pathogenic relatedness of X. oryzae pv. oryzae populations from diverse geographic areas in Bangladesh.     

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     

Elevational Variation in Diversity of Xanthomonas oryzae pv. oryzae in South‐West China

Virulence analysis and two polymerase chain reaction–based assays were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae (Xoo) from different elevations ranging from 150 to 2600 m in south‐west China. Among the 218 isolates of Xoo, 18 pathotypes were identified using six near‐isogenic rice lines, each containing a single resistance gene. Among them, pathotype 9 predominated in low and mid‐elevations was virulent to all resistance genes, including Xa2, Xa3, xa5, xa13, Xa14 and Xa18. However, pathotype 2 was predominant at high elevation and was virulent to Xa18 only. The 18 pathotypes were grouped into four clusters. Isolates belonging to cluster 1 were mainly found at high and mid‐elevations, while those of cluster 4 were mainly found at low elevations. There were significant trends of virulence of isolates from low to high with the elevation from high to low. The ERIC and J3 primers were used to screen the genomes of 218 isolates, and 56 molecular haplotypes were found. Multiple correspondence analyses revealed that 56 haplotypes were divided into four putative genetic lineages. Lineage 2 was the most frequently detected from 150 to 2600 m; it was clearly shown that isolates from high elevation with 80% is much more than from low and mid‐elevation in the lineage. It is intriguing that genetic variation of Xoo is restricted by physical geographical barriers of elevations. This is the first report on the relationship of pathotypic and genotypic diversity of Xoo at different elevations.     

Salicylic acid-induced accumulation of biochemical components associated with resistance against Xanthomonas oryzae pv. oryzae in rice

Seed treatment and foliar sprays of salicylic acid (SA) provided protection in rice against bacterial leaf blight (BLB) caused by bacterial Xanthomonas oryzae pv. oryzae (Xoo). Treatment of rice with exogenous SA reduced disease severity by more than 38%. Superoxide anion production and hypersensitive response increased approximately 28% and 110% at 6 and 48?h after Xoo inoculation, respectively, for plants treated with SA. Moreover, the Xoo in treated rice plants grew more slowly, resulting in a population that was half of that observed in the control. Fourier transform infrared spectroscopy analysis revealed that the higher ratios of 1233/1517, 1467/1517, and 1735/1517?cm^?1 observed in treated rice suggested alteration of monomer composition of lignin and pectin in the rice cell wall. Exogenous SA-treated rice had more amide I β-sheet structure and lipids as shown by the peaks at 1629, 2851, and 1735?cm^?1. These biochemical changes of rice treated with SA and inoculated with Xoo were related to primed resistance of the rice plants to BLB disease.     

A "defeated" rice resistance gene acts as a QTL against a virulent strain of Xanthomonas oryzae pv. oryzae

The genetic components responsible for qualitative and quantitative resistance of rice plants to three strains (CR4, CXO8, and CR6) of Xanthomonas oryzae pv. oryzae (Xoo) were investigated using a set of 315 recombinant inbred lines (RILs) from the cross Lemont (japonica) × Teqing (indica) and a complete linkage map with 182 well distributed RFLP markers. We mapped a major gene (Xa4) and ten quantitative trait loci (QTLs) which were largely responsible for segregation of the resistance phenotype in the RILs. The Teqing allele at the Xa4 locus, Xa4 ^T , acted as a dominant resistance gene against CR4 and CXO8. The breakdown of Xa4 ^T -associated resistance mediated by the mutant allele at the avrXa4 locus in the virulent strain CR6 results from significant changes in both gene action (lose of dominance) and the magnitude of gene effect (≈50% reduction). Nevertheless, Xa4 ^T still acted as a recessive QTL with a significant residual effect against CR6. The mutant alleles at the avrXa4 locus in CXO8 and CR6 that lead to a reduction in effect, or “breakdown”, of Xa4 ^T were apparently accompanied by corresponding penalties for their fitness. The quantitative component of resistance to Xoo in the RILs was largely due to a number of resistance QTLs. Most resistance QTLs mapped to genomic locations where major resistance genes and/or QTLs for resistance to Xoo, blast and sheath blight were identified in the same cross. Most QTLs showed consistent levels of resistance against all three Xoo strains. Our results suggest that a high level of durable resistance to Xoo may be achieved by the cumulative effects of multiple QTLs, including the residual effects of “defeated” major resistance genes. Received: 28 April 1998 / Accepted: 9 October 1998     

Genotypic and Pathotypic Diversity of Xanthomonas oryzae pv. oryzae Strains in Taiwan

Rice bacterial leaf blight, caused by Xanthomonas oryzae pv. oryzae [(Ishiyama) Swings et al. 1990] (Xoo), is a major rice disease of the second crop season in Taiwan. A total of 88 Xoo strains collected from 10 major rice cultivating areas in Taiwan from 1986, 1997, 2000, 2004, and 2011 were characterized by repetitive‐element PCR (REP‐PCR) fingerprinting and virulence analyses. Among the five genetic clusters identified by the pJEL1/pJEL2 (IS1112‐based) and REP1R‐Dt/REP2‐D [repetitive extragenic palindromic (REP)‐based] primer sets, clusters A, C and D contained Xoo strains from geographically distant regions, which suggests a high frequency of Xoo dispersal in Taiwan. The 88 Xoo strains were evaluated by inoculations on IRBB near‐isogenic lines and five Taiwan rice cultivars. A subset of 45 moderately or highly virulent strains were classified into 15 pathotypes by their compatible or incompatible reactions on IR24 and 12 IRBB near‐isogenic lines, each containing a single resistance gene. Analysis of molecular haplotypes and pathotypes revealed a partial relationship. IRBB5, IRBB21 and IRBB4 were incompatible with 96%, 96% and 73% of the strains, so xa5, Xa21 and Xa4 can recognize most of the Xoo strains in Taiwan and elicit resistance. In contrast, IRBB3 (Xa3), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11), IRBB13 (xa13) and IRBB14 (Xa14) were susceptible to almost all of the 45 Xoo strains. Inoculation trials revealed significant differences in the susceptibility of five Taiwan cultivars to Xoo (from high to low susceptibility: Taichung Sen 10 >  IR24, Taichung Native 1 >  Taichung 192, Taikeng 9, Tainan 11). This study provides useful information for resistance breeding and the development of disease management strategies against bacterial blight disease of rice.     

A “defeated” rice resistance gene acts as a QTL against a virulent strain of Xanthomonas oryzae pv. oryzae

The genetic components responsible for qualitative and quantitative resistance of rice plants to three strains (CR4, CXO8, and CR6) of Xanthomonas oryzae pv. oryzae (Xoo) were investigated using a set of 315 recombinant inbred lines (RILs) from the cross Lemont (japonica) × Teqing (indica) and a complete linkage map with 182 well distributed RFLP markers. We mapped a major gene (Xa4) and ten quantitative trait loci (QTLs) which were largely responsible for segregation of the resistance phenotype in the RILs. The Teqing allele at the Xa4 locus, Xa4 ^T , acted as a dominant resistance gene against CR4 and CXO8. The breakdown of Xa4 ^T -associated resistance mediated by the mutant allele at the avrXa4 locus in the virulent strain CR6 results from significant changes in both gene action (lose of dominance) and the magnitude of gene effect (≈50% reduction). Nevertheless, Xa4 ^T still acted as a recessive QTL with a significant residual effect against CR6. The mutant alleles at the avrXa4 locus in CXO8 and CR6 that lead to a reduction in effect, or “breakdown”, of Xa4 ^T were apparently accompanied by corresponding penalties for their fitness. The quantitative component of resistance to Xoo in the RILs was largely due to a number of resistance QTLs. Most resistance QTLs mapped to genomic locations where major resistance genes and/or QTLs for resistance to Xoo, blast and sheath blight were identified in the same cross. Most QTLs showed consistent levels of resistance against all three Xoo strains. Our results suggest that a high level of durable resistance to Xoo may be achieved by the cumulative effects of multiple QTLs, including the residual effects of “defeated” major resistance genes.     

Bacterial leaf blight resistance genes (Xa21, xa13 and xa5) pyramiding through molecular marker assisted selection into rice cultivars

Marker assisted selection was employed to pyramid three bacterial blight resistance genes Xa21, xa13 and xa5 into high yielding susceptible rice cultivars ADT43 and ADT47. With the assistance of PCR markers, homozygous and heterozygous genotypes were identified in F₂ generation of two crosses (ADT43 × IRBB60 and ADT47 × IRBB60) and goodness of fit was tested. Eighty nine plants from F₃ generation of ADT43 × IRBB60 were also screened for resistance genes. The genotypes carrying resistance genes in different combinations were identified. The pyramided lines showed a wider spectrum and higher level of resistance against two Xoo isolates under field conditions.     

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.     

Dissection of the factors affecting development-controlled and race-specific disease resistance conferred by leucine-rich repeat receptor kinase-type <Emphasis Type="Italic">R</Emphasis> genes in rice

Development-controlled resistance and resistance specificity frequently restrict the application of a disease resistance (R) gene in crop breeding programs. Xa3/Xa26 and Xa21, encoding leucine-rich repeat (LRR)-kinase type plasma membrane proteins, mediate race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), which causes bacterial blight, one of the most devastating rice diseases. Plants carrying Xa3/Xa26 and plants carrying Xa21 have different resistance spectra and the functions of the two R genes are regulated by developmental stage. Four chimeric genes encoding proteins consisting of different parts of XA3/XA26 and XA21 were constructed by domain swapping and transformed into a susceptible rice variety. The resistance spectra and development-regulated resistance of the transgenic plants carrying Xa3/Xa26, Xa21, or chimeric gene to different Xoo strains were analyzed in the same genetic background. The results suggest that the gradually increased expression of Xa3/Xa26 and Xa21 plays an important role in the progressively enhanced Xoo resistance during rice development. In addition, the LRR domains of XA3/XA26 and XA21 are important determinants of race-specific recognition during rice–Xoo interaction, but juxtamembrane regions of the two R proteins also appear to contribute to resistance specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dissection of the genetic architecture of rice resistance to Xanthomonas oryzae pv. oryzae using a genomewide association study

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), usually causes serious rice yield loss in many countries. Rice breeders have used resistance (R) genes to control the disease but many of the resistant cultivars become susceptible few years after releasing. Identification of new R genes to Xoo is one of the main objectives in rice breeding programs. In this study, we used a genomewide association study (GWAS) to analyse the resistance against the Xoo race C1 using the Rice Diversity Panel 1 (RDP1). Disease evaluation of the RDP1 population to C1 indicated that the AUS subgroup conferred a higher level of resistance to C1 than other subgroups. Genomewide association mapping identified 15 QTLs that are distributed on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, 10 and 12. Some of them are located in the regions without known resistance loci or QTLs. This study demonstrated the effectiveness of GWAS on the genetic dissection of rice resistance to Xoo and provided many Xoo resistance‐associated SNP markers for rice breeding.     

Transgenic expression of the rice Xa21 pattern‐recognition receptor in banana (Musa sp.) confers resistance to Xanthomonas campestris pv. musacearum

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in east and central Africa. The spread of BXW threatens the livelihood of millions of African farmers who depend on banana for food security and income. There are no commercial chemicals, biocontrol agents or resistant cultivars available to control BXW. Here, we take advantage of the robust resistance conferred by the rice pattern‐recognition receptor (PRR), XA21, to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). We identified a set of genes required for activation of Xa21‐mediated immunity (rax) that were conserved in both Xoo and Xcm. Based on the conservation, we hypothesized that intergeneric transfer of Xa21 would confer resistance to Xcm. We evaluated 25 transgenic lines of the banana cultivar ‘Gonja manjaya’ (AAB) using a rapid bioassay and 12 transgenic lines in the glasshouse for resistance against Xcm. About 50% of the transgenic lines showed complete resistance to Xcm in both assays. In contrast, all of the nontransgenic control plants showed severe symptoms that progressed to complete wilting. These results indicate that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance against Xcm. Furthermore, this work demonstrates the feasibility of PRR gene transfer between monocotyledonous species and provides a valuable new tool for controlling the BXW pandemic of banana, a staple food for 100 million people in east Africa.     

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.     

Isolation and characterization of rice mutants compromised in<Emphasis Type="Italic"> Xa21</Emphasis>-mediated resistance to<Emphasis Type="Italic"> X. oryzae</Emphasis> pv.<Emphasis Type="Italic"> oryzae</Emphasis>

The rice gene, Xa21, confers resistance to diverse races of Xanthomonas oryzae pv. oryzae (Xoo) and encodes a receptor-like kinase with leucine-rich repeats in the extra-cellular domain. To identify genes essential for the function of the Xa21 gene, 4,500 IRBB21 (Xa21 isogenic line in IR24 background) mutants, induced by diepoxybutane and fast neutrons, were screened against Philippine race six (PR6) Xoo for a change from resistance to susceptibility. From two greenhouse screens, 23 mutants were identified that had changed from resistant to fully (6) or partially (17) susceptible to PR6. All fully susceptible mutants carried rearrangements at the Xa21 locus as detected by PCR and Southern hybridization. For the partially susceptible mutants, no changes were detected at the Xa21 locus based on Southern and PCR analyses. However, two of these mutants were confirmed via genetic analysis to have mutations at the Xa21 locus. Partially susceptible mutants exhibited variation in level of susceptibility to different Xoo strains, suggesting that they may carry different mutations required for the Xa21-mediated resistance. The mutants identified in this study provide useful materials for dissecting the Xa21-mediated resistance pathway in rice.Communicated by D.J. Mackill     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.