Xoo Isolates Research Articles

Development and Validation of an SNP Marker for Identifying Xanthomonas oryzae pv. oryzae Thai Isolates That Break xa5-Mediated Bacterial Blight Resistance in Rice.

Xanthomonas oryzae pv. oryzae (Xoo) is a pathogenic bacterium responsible for bacterial blight (BB) disease in rice, primarily mediated by the interaction between the plant and pathogen. The virulence mechanism involves the activation of the Sugars Will Eventually be Exported Transporter (SWEET) gene family in rice by transcription activator-like effectors derived from Xoo. The BB resistance gene xa5 has been identified as one of the most effective genes against Thai Xoo isolates, but xa5-mediated resistance-breaking Xoo strains have emerged. This study aimed to develop a single nucleotide polymorphism (SNP) marker for precise identification of xa5-mediated resistance-breaking Xoo. Comparative genomics of Thai Xoo isolates Xoo16PK001 and Xoo16PK002, which were incompatible and compatible with rice variety IRBB5 carrying xa5, respectively, identified eight SNP positions for the development of an SNP marker. The SNP marker XooE6 yields a specific 1,143 bp PCR product unique to Xoo16PK002. Screening 61 Thai isolates using XooE6 identified two positives: Xoo20PL010 and Xoo20UT002. Inoculation tests on rice varieties IRBB5 and IRBB13 demonstrated compatibility with IRBB5 and incompatibility with IRBB13, which bears Xa5 and xa13. Xoo16PK001 (XooE6-negative) showed different virulence. Inoculation on IRBB21 harboring Xa5, Xa13, and Xa21 resulted in partial resistance to both XooE6-positive and -negative strains. XooE6-positive strains up-regulated SWEET11 and suppressed SWEET14 in IRBB5, while Xoo16PK001 slightly induced SWEET11 but activated SWEET14 in IRBB13. This highlights the potential of XooE6 to identify xa5-mediated resistance-breaking Xoo strains and elucidate their pathogenic mechanisms through the upregulation of SWEET11.

In planta ANALYSIS OF SALICYLIC ACID MEDIATED MANAGEMENT OF

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a catastrophic bacterial disease of rice, and researchers are facing difficulties in developing the management practices, whereas the continued use of bactericides viz, streptomycin, copper oxychloride leads to the development of resistant Xoo strains. As a consequence, the development of an environmentally safe bactericide for the management of this devastating disease is needed. The present study focused on identification of effective synthetic activators against bacterial leaf blight in rice during their interaction. Survey was conducted to collect different isolates of Xoo and their morphological characters and biochemical properties were determined. All the 10 isolates exhibited circular to flattened slimy yellow colour colonies of the pathogen and showed positive reaction for citrate utilization, lysine utilization and ornithine utilization test. All the ten isolates showed negative reaction for sorbitol utilization. The collected isolates were subjected to PCR for the amplification of 16S-23S intergenic region with the species specific primer and obtained amplicon at ⁓470 bp for the confirmation at molecular level. The virulent isolate was identified by measuring biofilm formation and xanthomonadin pigment which revealed that the isolate ADT 53 strain T3 with the accession number OR121377 recorded maximum OD value of 0.356 for biofilm formation at 570 nm and 0.686 for xanthomonadin pigment at 445 nm. In planta studies revealed that salicylic acid at 100 ppm has the maximum inhibitory effect against bacterial leaf blight disease with the minimal lesion size of 0.7 cm compared to negative control (water). Keywords: Activators, BLB, Biofilm, Characterization, in planta, Xanthomonadin.

Insights into the virulence profiles and molecular diversity of Xanthomonas oryzae pv. oryzae isolates associated with bacterial blight of rice in major districts of Karnataka, India

Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating disease affecting rice worldwide. In this study, to characterize Xoo isolates and evaluate the effectiveness of various BLB-resistant genes in rice, a set of 29 rice differentials each carrying, single or combinations of BLB resistant genes were used for characterizing Xoo isolates under glasshouse condition. None of the tested isolates were able to show complete virulence across all Near Isogenic Lines (NILs). All isolates varied with degrees of resistance and susceptibility. Notably, isolate KXo-3 exhibited high virulence. Among single BLB resistant genes, IRBB-8 (xa8) and IRBB-13 (xa13) were found highly promising while among two gene combinations, IRBB-51 (Xa4+xa13), IRBB-52 (Xa4+Xa21) and IRBB-53 (xa5+xa13). Among three and four gene combination of BB resistant genes, IRBB-56 (Xa4+xa5+xa13), IRBB-57 (Xa4+xa5+Xa21), IRBB-58 (Xa4+xa13+Xa21), IRBB-59 (Xa5+xa13+Xa21), IRBB-60 (Xa4+xa5+xa13+Xa21), IRBB-63 (xa5+Xa7+xa13) and IRBB-65 (Xa4+Xa7+xa13+Xa21) were resistant against almost all the Xoo isolates. Based on disease reactions, Xoo isolates were grouped into 6 pathotypes, as IXoPt # 1, 2, 4, 5, 17 and 22. IXoPt-22 was highly virulent and IXoPt-1 was least virulent. IXoPt-2 was widely distributed with the maximum distribution frequency of 23.8. Molecular diversity using IS1112 repetitive elements showed that the Xoo isolates were genetically very diverse with three major clusters, I, II and III. The haplotype diversity resulted in 27 haplotypes generation. The isolates of Belagavi, Haveri and Davanagere were very diverse with distinct haplotypes. These findings enhance our understanding of BLB resistance mechanisms and Xoo population diversity, aiding in the development of effective disease management strategies in rice cultivation.

Broad-Spectrum Resistance and Monogenic Inheritance of Bacterial Blight Resistance in an Indigenous Upland Rice Germplasm ULR207

Bacterial blight (BB) caused by Xanthomonas oryzae pv. Oryzae (Xoo) is a serious disease of rice worldwide that can reduce crop yield and affect food insecurity. A rice resistance variety is an alternate way to solve this problem. The broad-spectrum resistance (BSR) of ULR207 is important for durable resistance to several of the Xoo isolates. However, the inheritance of this resistance gene in ULR207 must be known before it can be utilized. Thus, this study aimed to survey the BB resistance gene with reference to the BB resistance gene for identification of non-analogous or analogous genes and confirmation of a broad-spectrum resistance, to investigate the gene effect, the number of genes, and the heritability of the BB resistance gene in the ULR207 variety. Six populations of two crosses (Maled Phai × ULR207 and RD6 × ULR207), i.e., ULR207 (Donor parent), Maled Phai and RD6 (Recurrent parent), F1, F2, BC1P1, and BC1P2 were constructed. These materials were evaluated for BB resistance by clipping methods under greenhouse conditions using a virulence isolate of a pathogen in Thailand. The results showed that ULR207 exhibited the strongest against BB with 0.8 of BSR with low area under the disease progress curve (AUDPC). Molecular screening for surveying of the BB resistance gene in ULR207 revealed a non-analogous resistance gene with resistance check varieties. The phenotype of the disease lesion length of F2 and BC1P2 populations exhibited a ratio of 1:3 and 1:1 (resistant: susceptible), respectively, revealing a single recessive gene in both crosses. The scaling test parameters A, B, and C were non-significant (p < 0.01), indicating that variation in data was sufficiently explained by additive (d) and dominance (h) components. The gene action of ULR207 was controlled by additive gene action. Heritability of the two crosses, Maled Phai x ULR207 and RD6 x ULR207, exhibited high values with 0.817 and 0.716, whereas the numbers of the genes were 1.4 and 1.2, respectively. The result indicated that the breeding strategy could be employed in early generations when using ULR207 as a new source of bacterial blight resistance.

Screening of Rice Genotypes for Bacterial Blight of Rice Under Artificial Inoculation Method and Monitoring Field Virulence of Xanthomonas oryzae pv. oryzae

The present study was conducted at the main rice research centre, Navsari Agricultural University, Navsari, Gujarat, India during kharif (July–November, 2020) to study the screening of rice genotypes for bacterial blight of rice under artificial inoculation method and monitoring field virulence of Xanthomonas oryzae pv. oryzae. Thirty genotypes along with two susceptible checks against bacterial blight pathogen under artificial inoculation condition revealed that, 3 genotypes were found moderately resistant viz., NVSR-466, NVSR-396 and ISM. Whereas, 12 genotypes viz., NVSR-411, NVSR-443, NVSR-447, NVSR-453, GNR-6, IR-28, NAUR-1, GNR-2, Gurjari, GNR-4, Krishna kamod and GAR-13 were found susceptible to Xoo and two checks viz., GR-11 and TN-1 were found highly susceptible. Observing to the disease severity, by screening 29 IRBB differentials and two checks employed for research on monitoring virulence of Xoo. Out of 29 differentials, 3 entries were found resistant viz., IRBB-53, IRBB-57 and IRBB-60. Whereas, 3 entries viz., IRBB-50, IRBB-52 and IRBB-61 were moderately susceptible reaction and all the single genes possessing entries were found susceptible to Navsari isolates of Xoo. The goal of this study provided valuable insights to determine the potential management strategies for the disease in rice crops by employing methodology of two rows of 2 meters in length for each entry, the susceptible check TN-1 and GR-11 were planted after the 10th test entry for screening of rice genotypes and monitoring the field virulence of Xoo.

Genome Sequence Resource for Xanthomonas oryzae pv. oryzae (Causal Agent of Bacterial Leaf Blight of Rice) Isolated from Pakistan

Bacterial leaf blight, one of the most destructive diseases of rice, is prevalent in the major rice-growing regions of the world. Bacterial leaf blight is caused by Xanthomonas oryzae pv. oryzae ( Xoo) and greatly affects the quantity and quality of the rice crop. We sequenced the whole genome of Xoo isolated from a Basmati rice-growing region of Pakistan using long-read Oxford Nanopore sequencing technology. About 637 MB of data was retrieved, corresponding to 133× genome sequencing coverage. The final assembly yields a circular chromosome of 4,930,682 bp. The complete genome of the Xoo isolate from Pakistan will enhance sequence resources for global comparison of Xoo diversity across the region. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Survey and Isolation of Rice Bacterial Leaf Blight Caused by Xanthomonas oryzae pv. oryzae

A survey was conducted at different rice growing regions of Tamil Nadu such as Erode, Tanjore and Coimbatore. Among the areas surveyed the maximum disease incidence was recorded at Tanjore in the rice variety ADT 54 with the percent disease incidence of 50.23%. Initially one bacterial pathogen Xanthomonas oryzae pv.oryzae was isolated in nutrient agar medium from the leaves having characteristic symptom. Totally 10 isolates of Xoo were collected from three different regions of Tamil Nadu. On isolating the pathogen the colonies are found to be well separated with slimy yellow color and mucoid growth.

Tal6b/AvrXa27A, a hidden TALE targeting the susceptibility gene OsSWEET11a and the resistance gene Xa27 in rice

Xanthomonas oryzae pv. oryzae (Xoo) secretes transcription activator-like effectors (TALEs) to activate rice susceptibility (S) genes, causing bacterial blight (BB), as well as resistance (R) genes, leading to defense against BB. This activation follows a gene-for-gene paradigm that results in an arms race between the TALE of the pathogen and effector-binding elements (EBEs) in the promoters of host genes. In this study, we characterized a novel TALE, designated Tal6b/AvrXa27A, that activates the rice S gene OsSWEET11a and the rice R gene Xa27. Tal6b/AvrXa27A is a member of the AvrXa27/TalAO class and contains 16 repeat variable diresidues (RVDs); one RVD is altered and one is deleted in Tal6b/AvrXa27A compared with AvrXa27, a known avirulence (avr) effector of Xa27. Tal6b/AvrXa27A can transcriptionally activate the expression of Xa27 and OsSWEET11a via EBEs in their corresponding promoters, leading to effector-triggered immunity and susceptibility, respectively. The 16 RVDs in Tal6b/AvrXa27A have no obvious similarity to the 24 RVDs in the effector PthXo1, but EBETal6b and EBEPthXo1 are overlapped in the OsSWEET11a promoter. Tal6b/AvrXa27A is prevalent among Asian Xoo isolates, but PthXo1 has only been reported in the Philippine strain PXO99A. Genome editing of EBETal6b in the OsSWEET11a promoter further confirmed the requirement for OsSWEET11a expression in Tal6b/AvrXa27A-dependent susceptibility to Xoo. Moreover, Tal6b/AvrXa27A resulted in higher transcription of Xa27 than of OsSWEET11a, which led to a strong, rapid resistance response that blocked disease development. These findings suggest that Tal6b/AvrXa27A has a dual function: triggering resistance by activating Xa27 gene expression as an avirulence factor and inducing transcription of the S gene OsSWEET11a, resulting in virulence. Intriguingly, Tal6b/AvrXa27A, but not AvrXa27, can bind to the promoter of OsSWEET11a. The underlying recognition mechanism for this binding remains unclear but appears to deviate from the currently accepted TALE code.

Pathotyping and Virulence Analysis of Xanthomonas oryzae pv. oryzae Causing Bacterial Blight of Rice in Tamil Nadu

Background-Bacterial leaf blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases affecting rice production worldwide. Resistance breeding is considered a durable, effective, and eco-friendly approach to control BB. Several BB resistance (R) genes have been identified that confer resistance against various strains of Xoo. The dynamic nature of this pathogen needs proper knowledge of pathotype composition and its virulence among the diverse Xoo strains which is imperative for designing a resistance breeding program. Methods-In this study, we conducted an extensive survey to collect BB infected samples from diverse rice-growing regions of Tamil Nadu. Further, the samples were identified as Xoo through morphological and molecular identification methods. The pathotyping and virulence profiling of Xoo isolates was carried out on a set of rice differentials consisting of 22 near-isogenic lines (NILs) of IR24 possessing different BB resistance genes under glasshouse conditions during Kharif 2021-2022. Results-Based on the disease reaction in 22 rice BB differentials (IRBB lines), the Xoo isolates were grouped into six pathotypes (I II, III, IV, V, and VI). Further, the virulence analysis revealed that the near-isogenic lines possessing single R gene and gene pyramids were susceptible to about 20-88% and 4-20% respectively to Xoo isolates. R genes and their pyramids such as (Xa7, Xa21, Xa4+xa5+Xa21, Xa4+xa5+xa13 Xa4+xa13+Xa21, xa5+xa13+Xa21, and Xa4+Xa21+xa5+Xa21) were found to be effective against all the pathotypes in Tamil Nadu. The pathotype III was predominantly distributed (40%) over the Tamil Nadu. The isolates found in the pathotypes I and II are more virulent and in the pathotypes V, VI are less virulent. The current study identified the virulence nature of diverse Xoo isolates and the effectiveness of resistance (R) genes against the Xoo isolates. Therefore, the effective R genes identified in this study could be an effective source for resistance breeding program against BB in Tamil Nadu.

T3SS mediated transcriptional reprogramming of rice by the virulent Indian race 4 of Xanthomonas oryzae pv. oryzae

Bacterial blight (BB) incited by Xanthomonas oryzae pv. oryzae (Xoo) is a constant threat to global rice production. Xoo attenuates host defense by directly translocating type III effectors (T3Es) into rice cytosol. The fleeting emergence of new virulent Xoo isolates and subsequent breakdown of resistance in the realm of an erratic climate entails the exploration of befitted resistance to prevent super-races of Xoo from becoming widespread. A comprehensive knowledge of T3Es-mediated transcriptional reprogramming of rice genes in response to Xoo infection remains elusive. Evidently, RNA seq-based transcriptome profiling of rice infected with an Indian virulent Xoo strain Race 4 relative to its T3SS-defective mutant strain (Xoo DhrpX) at early and late hours of bacterial infection yielded an array of differentially expressed genes (DEGs), 49 genes of which were selected for RT-qPCR analysis. The results revealed T3SS-dependent novel defence-related genes belonging to Nucleotide-binding leucine-rich repeat (NLRs), Receptor-like kinases (RLKs), PR proteins, enzymes, cytochrome P450 and secondary metabolites. These genes are direct targets for enhancing BB resistance as well silencing of susceptible targets through gene editing yields durable resistance.

Insights into the Diversity of Transcription Activator-Like Effectors in Indian Pathotype Strains of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a major rice pathogen, and its genome harbors extensive inter-strain and inter-lineage variations. The emergence of highly virulent pathotypes of Xoo that can overcome major resistance (R) genes deployed in rice breeding programs is a grave threat to rice cultivation. The present study reports on a long-read Oxford nanopore-based complete genomic investigation of Xoo isolates from 11 pathotypes that are reported based on their reaction toward 10 R genes. The investigation revealed remarkable variation in the genome structure in the strains belonging to different pathotypes. Furthermore, transcription activator-like effector (TALE) proteins secreted by the type III secretion system display marked variation in content, genomic location, classes, and DNA-binding domain. We also found the association of tal genes in the vicinity of regions with genome structural variations. Furthermore, in silico analysis of the genome-wide rice targets of TALEs allowed us to understand the emergence of pathotypes compatible with major R genes. Long-read, cost-effective sequencing technologies such as nanopore can be a game changer in the surveillance of major and emerging pathotypes. The resource and findings will be invaluable in the management of Xoo and in appropriate deployment of R genes in rice breeding programs.

Population genomics and pathotypic evaluation of the bacterial leaf blight pathogen of rice reveals rapid evolutionary dynamics of a plant pathogen.

The Xanthomonas oryzae pv. oryzae (Xoo) is a bacterial pathogen causing bacterial blight disease in rice, resulting in significant yield reductions of up to 50% in rice production. Despite its serious threat to food production globally, knowledge of its population structure and virulence evolution is relatively limited. In this study, we employed whole-genome sequencing to explore the diversity and evolution of Xoo in the main rice-growing areas of China over the past 30 years. Using phylogenomic analysis, we revealed six lineages. CX-1 and CX-2 primarily contained Xoo isolates from South China, while CX-3 represented Xoo isolates from North China. Xoo isolates belonging to CX-5 and CX-6 were the most prevalent across all studied areas, persisting as dominant lineages for several decades. Recent sporadic disease outbreaks were primarily caused by Xoo isolates derived from the two major lineages, CX-5 and CX-6, although Xoo isolates from other lineages also contributed to these outbreaks. The lineage and sub-lineage distributions of Xoo isolates were strongly correlated with their geographical origin, which was found to be mainly determined by the planting of the two major rice subspecies, indica and japonica. Moreover, large-scale virulence testing was conducted to evaluate the diversity of pathogenicity for Xoo. We found rapid virulence evolution against rice, and its determinant factors included the genetic background of Xoo, rice resistance genes, and planting environment of rice. This study provides an excellent model for understanding the evolution and dynamics of plant pathogens in the context of their interactions with their hosts, which are shaped by a combination of geographical conditions and farming practices. The findings of this study may have important implications for the development of effective strategies for disease management and crop protection in rice production systems.

Genome-Wide Association Study Using Genotyping by Sequencing for Bacterial Leaf Blight Resistance Loci in Local Thai Indica Rice

Bacterial leaf blight (BLB) is a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), which poses a significant threat to global rice production. In this study, a genome-wide association study (GWAS) was conducted using the genotyping-by-sequencing (GBS) approach to identify candidate single nucleotide polymorphisms (SNPs) associated with BLB resistance genes. The study utilized 200 indica rice accessions inoculated with seven distinct Xoo isolates and filtered highly significant SNPs using a minor allele frequency (MAF) of >5% and a call rate of 75%. Four statistical models were used to explore potential SNPs associated with BLB resistance, resulting in the identification of 32 significant SNPs on chromosomes 1–8 and 12 in the rice genome. Additionally, 179 genes were located within ±100 kb of the SNP region, of which 49 were selected as candidate genes based on their known functions in plant defense mechanisms. Several candidate genes were identified, including two genes in the same linkage disequilibrium (LD) decay as the well-known BLB resistance gene (Xa1). These findings represent a valuable resource for conducting further functional studies and developing novel breeding strategies to enhance the crop’s resistance to this disease.

Distribution of Xanthomonas oryzae pv. oryzae Pathotypes in Basmati-Rice-Growing Areas of Jammu and Kashmir, India

Rice is an important staple food for more than half of the world’s population. Though the genetic potential of commonly cultivated varieties of rice is diminished due to various biotic and abiotic constraints, bacterial leaf blight (BLB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is considered one of its most destructive diseases in India. Based on morpho-cultural characteristics, bacterial pathogens isolated from the leaves of a rice plant showing typical BLB symptoms were identified as Xanthomonas oryzae pv. oryzae. Morphological studies revealed that the pathogen is Gram-negative, a short rod, with rounded ends, single or in pairs, light yellow, circular, whitish yellow to straw-colored, convex, yellow, slightly raised, motile with a single polar flagellum, capsulate and non-spore-forming. Biochemical tests, viz., the Gram reaction, KOH test and catalyst test, showed a positive reaction for all the isolates. Twenty isolates of Xoo were collected from the basmati-growing areas of the Jammu, Samba and Kathua districts in the Jammu sub-tropics during 2019, and their pathogenicity was confirmed on five susceptible rice cultivars, viz., Basmati-370, Pusa-1121, TN-1, SJR and Jaya, by the leaf-clipping method, and subsequently, Koch’s postulate was established in each case. Seven Xoo pathotypes, viz., Pathotype 1, Pathotype 2, Pathotype 3, Pathotype 4, Pathotype 5, Pathotype 6 and Pathotype 7, were identified from the total sample of 20 isolates. Pathotype 2 was the most dominant (100%), followed by Pathotype 5 (44.44%), Pathotype 4 (40%), Pathotype 6 (40%), Pathotype 7 (33.33%), Pathotype 3 (22.22%) and Pathotype 1 (20%), in the Jammu sub-tropics. In Jammu district, Pathotype 5 was highly distributed (44.44%) followed by Pathotype 7 (33.33%) and Pathotype 3 (22.22%). Pathotype 4 and Pathotype 6 each showed a 40 percent distribution in Kathua district, followed by Pathotype 1 (20%). Only one pathotype, i.e., Pathotype 2, was recorded in Samba district with a 100 percent distribution. Five genes, viz., Xa13, Xa4, Xa13 and Xa5 + Xa13, showed complete resistance, whereas Xa4, Xa5, Xa7, Xa8, Xa21, Xa4 + Xa5 and Xa4 + Xa21 showed susceptible response against the test isolates. It was observed that most of the single BLB-resistant genes were moderately to highly susceptible to almost all the Xoo isolates, whereas combinations of BLB resistance genes possessed high resistance against all the Xoo isolates. The studies revealed that diverse pathogenic variations existed in the Xoo population in the basmati-growing region of Jammu and Kashmir. Based on the response exhibited by Xoo isolates on differential lines, seven pathotypes (Pathotype 1–7) were identified, and their virulence spectrum on rice differentials showed the occurrence of 5, 3, 10, 10, 20, 10 and 15 percent, respectively, in the Jammu sub-tropics. To develop durable and sustainable resistant cultivars, it is essential to identify predominate race(s) in a specific geographical area and continuously monitor the virulence pattern there.

Pathogenicity structure of the Xanthomonas oryzae pv. oryzae population causing rice bacterial blight in Vietnam

The rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) bacteria is rice’s most destructive disease. Due to the potential biomagnification caused by bactericides, deploying host resistance is the most effective method to protect the rice harvest. Studying the pathogenicity structure of the Xoo population helps propose the best breeding strategies to use the resistance gene pool. Among 12 resistance genes validated, xa5 and Xa21 maintained their efficacy against the Xoo population in Vietnam. Other genes, including Xa7 and xa13, remained effective in certain regions. The most destructive Xoo races distribute in the South, where intensive rice production was practiced. The same Xoo races found in different ecological regions illustrated the pathogen migration between adjacent areas. This manuscript proposed a designation system that converts the response of the differential set to each Xoo isolate into a binary, then into an octal system to name Xoo races appropriately.

Genetic diversity and pathotype profiling of Xanthomonas oryzae pv. oryzae isolates from diverse rice growing ecosystems of Karnataka state of India

Bacterial leaf blight (BLB) disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the significant constraints for rice production. A study was carried out to analyze the virulence, genetic diversity, and population structure of Xoo isolates collected from different rice ecosystems of Karnataka, India. The taxonomic identity of all 54 isolates was confirmed using a Xoo-specific PCR assay. The virulence assay on a set of twenty-eight near-isogenic differentials identified nine pathotype groups (KPthX-1 to KPthX-9). Among them, KPthX-6 was highly virulent, whereas KPthx-2 was less virulent. Differentials with four and five resistance (R) genes (Xa4+xa5+xa13+Xa21, Xa4+Xa7+xa13+Xa21, and Xa4+xa5+Xa7+xa13+Xa21) were effective against all the pathotypes. Further, genetic diversity was deduced using a universal marker set, JEL-1, and JEL-2, complementary to a conserved repetitive sequence IS1112. The amplicon size and numbers varied from 100 bp to 3 Kb and 1 to 25. Based on the amplicon counts, Jaccard’s co-efficient and phylogenetic analyses were carried out and categorized the 54 isolates into three clusters (I to III). Cluster-II contains the maximum number of isolates (27), followed by cluster-I (23 isolates) and cluster-III (one isolate). The structure analysis categorized the isolates into five subgroups, viz. SG1, SG2, SG3, SG4, and SG5 indicate the existence of five subpopulations. The present study has identified the genetic and virulence diversity of the Xoo population and reported the effective R gene/s for different rice ecosystems, which can be used in the breeding program for the eco-specific management of BLB of rice.

Pyramiding of Four Broad Spectrum Bacterial Blight Resistance Genes in Cross Breeds of Basmati Rice.

Pyramiding of major resistance (R) genes through marker-assisted selection (MAS) is a useful way to attain durable and broad-spectrum resistance against Xanthomonas oryzae pv. oryzae pathogen, the causal agent of bacterial blight (BB) disease in rice (Oryza sativa L.). The present study was designed to pyramid four broad spectrum BB-R genes (Xa4, xa5, xa13 and Xa21) in the background of Basmati-385, an indica rice cultivar with much sought-after qualitative and quantitative grain traits. The cultivar, however, is susceptible to BB and was therefore, crossed with IRBB59 which possesses R genes xa5, xa13 and Xa21, to attain broad and durable resistance. A total of 19 F1 plants were obtained, some of which were backcrossed with Basmati-385 and large number of BC1F1 plants were obtained. In BC1F2 generation, 31 phenotypically superior genotypes having morphological features of Basmati-385, were selected and advanced up to BC1F6 population. Sequence-tagged site (STS)-based MAS was carried out and phenotypic selection was made in each successive generation. In BC1F6 population, potentially homozygous recombinant inbred lines (RILs) from each line were selected and evaluated on the bases of STS evaluation and resistance to local Xanthomonas oryzae pv. oryzae (Xoo) isolates. Line 23 was found pyramided with all four BB-R genes i.e., Xa4, xa5, xa13 and Xa21. Five genotypes including line 8, line 16, line 21, line 27 and line 28 were identified as pyramided with three R genes, Xa4, xa5 and xa13. Pathological study showed that rice lines pyramided with quadruplet or triplet R genes showed the highest level of resistance compared to doublet or singlet R genes. Thus, line 23 with quadruplet, and lines 8, 16, 21, 27, and 28 with triplet R genes, are recommended for replicated yield and resistance trials before release as new rice varieties. Further, traditional breeding coupled with MAS, is a solid way to attain highly effective BB-resistant rice lines with no yield cost.

Functional characterization of the \(\textit{OsSWEET13 }\) promoter involved in the infection of \(\textit{ Xanthomonas oryzae }\) pv. \(\textit{oryzae (Xoo) }\) in rice cultivar TBR225

Xanthomonas oryzae pv. oryzae (Xoo) causes severe bacterial leaf blight (BLB) disease to many Vietnamese major rice cultivars, including the TBR225. OsSWEET13 belongs to group III of the OsSWEET gene family encoding sugar transport proteins, which is considered one of the “susceptibility” genes (S genes) necessary for BLB disease. In this study, the rice cultivar TBR225 was determined to be susceptible to 19/20 Vietnamese Xoo isolates collected from the Northern provinces. Two of the three tested isolates (VXO_60 and VXO_96 isolates) were shown to up-regulate OsSWEET13 upon the Xoo infection of the TBR225 cultivar. The TBR225 OsSWEET13 promoter was isolated for sequencing analysis. The isolated DNA fragment was 615 bp in size, contained an effector binding element (EBE) PthXo2 that was recognized by the type III-secretory transcription activator-like (TAL) proteins of the Xoo. This promoter showed a similarity of more than 99% to the published OsSWEET13 promoter sequences (AP014967.1 and CP018167.1). Our findings are basic for the generation of highyielding rice varieties with resistance to BLB disease by genetic engineering in Vietnam.

Phenotypic Broad Spectrum of Bacterial Blight Disease Resistance from Thai Indigenous Upland Rice Germplasms Implies Novel Genetic Resource for Breeding Program

Bacterial blight (BB) disease, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most devastating diseases of rice worldwide. Breeding for BB resistance has been utilized to overcome this constraint of rice production; however, limited genetic resources of BB resistance or non-desirable genetic linkage between BB disease resistance and agronomic traits have become major obstacles. Interestingly, indigenous upland rice cultivars cultivated across Thailand are considered to be novel genetic resources of BB resistance for rice cultivar improvement through breeding programs. In this study, we screened for BB disease resistance among 256 indigenous upland rice cultivars using individual inoculation of two virulent Xoo isolates; NY1-1 and MS1-2, under greenhouse conditions. The results showed that 19 indigenous rice cultivars demonstrated BB disease resistance abilities after inoculation. These 19 upland rice cultivars were further examined for broad-spectrum resistance (BSR) performance through five individual Xoo isolate inoculations, under greenhouse conditions. Moreover, a mixed five Xoo isolate inoculation, including NB7-8, CM3-1, CN2-1, MS1-2, and NY1-1, was conducted to assess the BSR for BB resistance of those 19 cultivars under field conditions. Simultaneously, independent plants of the 19 varieties were grown without inoculation in the field to observe the disease reactions from the natural infection caused by local Xoo isolates. The results of the three experiments herein showed that five indigenous upland rice cultivars—ULR024, ULR029, ULR172, ULR207, and ULR356—consistently expressed 100% BSR to BB disease, as well as the resistance check varieties, IRBB5. This further illustrated that ULR024, ULR029, ULR172, ULR207, and ULR356 upland rice cultivars were phenotypically resistant to five Xoo isolates, within those (a) inoculated with five individual isolates under greenhouse conditions or (b) inoculation with five mixed isolates under field conditions. Moreover, the ULR024, ULR029, ULR172, ULR207, and ULR356 revealed BB disease-resistance abilities to natural infection. These results present novel genetic resources from indigenous upland rice cultivars in further breeding program of BB resistance in varied rice cultivars.

Molecular characterization and screening of Xanthomonas oryzae pv. oryzae, isolated from Pakistan for prediction of bacterial leaf blight-resistant basmati rice

Bacterial leaf blight (BLB) is a devastating disease of rice caused by Xanthomonas oryzae pv.oryzae (Xoo), severely affects grain quality and reduces yield in all rice-growing areas of Pakistan. The pathogen is highly dynamic and quickly evolves itself when encountered with new resistant genes, hence, the knowledge about existing virulence range within the pathogen population and availability of corresponding resistance gene source is of immense importance. The present study focused on molecular dissection and evaluating the effectiveness of different resistance gene combinations against Xoo isolates collected from seven major rice-growing districts of Punjab Pakistan. Phenotypic characterization of collected Xoo isolates has shown that all of them were rod-shaped Gram-negative bacteria. However, oryzae species and oryzae pathovar were confirmed in only 22 out of 50 isolates using multiplex PCR. Analysis of loop-mediated isothermal amplification validated the lineage and close relatedness of Pakistani Xoo isolates with the already reported Asian Xoo population. Pathogenicity test on susceptible rice variety Super Basmati has shown that out of twenty-two tested Xoo isolates, four isolates from three different districts fell into the most virulent category with an average lesion length of ≥15 cm. Pathotyping of selected resistant rice lines against Xoo isolates distributed 22 isolates into nine pathotypes based on their interaction with R genes. Current molecular and phenotypic profiling of isolated Pakistani Xoo isolates may serve as a valuable reference for global comparison to manage BLB disease of rice in real-time.