Lactococcus lactis subsp. lactis as a biocontrol agent against phytopathogens causing rice bacterial leaf blight.

Abstract. Rahma H, Nurbailis, Busniah M, Kristina N, Larasati Y. 2021. The potential of endophytic bacteria to suppress bacterial leaf blight in rice plants. Biodiversitas 23: 775-782. Endophytic bacteria are potential as biocontrol agents against bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae in rice to support sustainable agriculture. This study aimed to select and characterize 22 endophytic bacteria isolated from healthy rice, determine their ability to promote plant growth and suppress bacterial leaf blight disease in rice and also identify potential endophytic bacterial isolates. The study was arranged in a Completely Randomized Design with 24 treatments and three repetitions. The treatments used in the current study consisted of Xanthomonas oryzae infected plants and treated with endophytic bacterial isolates; infected plants without endophytic bacteria treatment (positive control), non-infected plants (negative control). Identification of potential endophytic bacteria was performed based on 16S rRNA sequences. Three out of 22 bacterial isolates, i.e., LmB1, LmA6, and LmB2 were able to suppress bacterial leaf blight disease with severity levels of 35.82%, 23.78%, and 23.78%, respectively. Based on the rice plant growth parameters, three bacterial isolates (LmA6, LmB1, and LmB35) were able to increase the growth of rice plants with an average value of 69.56%, 56.51%, and 47.82%, respectively. Two bacterial isolates, i.e., LmB 1 and LmA6 suppress the development of bacterial leaf blight disease and increase the growth of rice plants. Based on DNA sequence comparisons of DNA fragments amplified by 16S rRNA related marker of the selected bacterial isolates and database, then LmA6, LmB2, LmB1, and LmB35 had similarities with Bacillus cereus MD152 (96.87%), Bacillus thuringiensis ATCC 10792 (98.20%), Ochrobactrum intermedium strain OI1 (97.52%), and Stenotrophomonas maltophilia strain A1w2 (97.92%), respectively. Our study revealed that the indigenous endophytic bacteria from rice plants could be potential biological agents for controlling bacterial leaf blight disease and increasing plant growth.

Bacterial leaf blight (BLB) disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases worldwide in rice-growing regions. The Ineffectiveness of chemicals in disease management has increased the interest in phage therapy. In this study, we isolated 19 bacteriophages, infecting Xoo, from a rice field, which belonged to phage families Siphoviridae, Myoviridae, and Podoviridae on the basis of electron microscopy. Among 19 phages, Phage vB_XooS_NR08, a member of the Siphoviridae family, expressed antibacterial activity against all Xoo strains tested and did not lyse X. campestris and other unrelated bacterial hosts. Phage NR08 showed more than 80% viability at a temperature range of 4°C-40°C, pH range of 5-9, and direct exposure to sunlight for 2 h, whereas UV light and chemical agents were highly detrimental. In a one-step growth curve, NR08 has a 40-min latent period, followed by a 30-min burst period with a burst size of 250 particle/bacterium. The genome of NR08 is double-stranded DNA, linear having a size of 98,812 bp with a G + C content of 52.9%. Annotation of the whole-genome sequence indicated that NR08 encodes 142 putative open reading frames (ORFs), including one ORF for tRNA, namely, trna1-GlnTTG. Comparative genome analysis of NR08 showed that it shares maximum similarity with Pseudomonas phage PaMx42 (40% query coverage, 95.39% identity, and acc. Length 43,225) and Xanthomonas phage Samson (40% query coverage, 96.68% identity, and acc. Length 43,314). The average alignment percentage (AP) of NR08 with other Xoophages was only 0.32 to 1.25 since the genome of NR08 (98.8 kb) is almost double of most of the previously reported Xoophages (43-47 kb), thus indicating NR08 a novel Xoophage. In in vitro bacterial challenge assay, NR08 showed bacteriostasis up to 24 h and a 99.95% reduction in bacterial growth in 48 h. In rice pot efficacy trials, single-dose treatment of NR08 showed a significant reduction in disease up to 90.23% and 79.27% on 7 and 21 dpi, respectively. However, treatment using 2% skim milk-supplemented phage preparation was significantly less effective as compared to the neat phage preparation. In summary, this study characterized a novel Xoophage having the potential as a biocontrol agent in the mitigation of BLB in rice.

Three plants extracts were used for the management of Xanthomonas oryzae pv. oryzae in vitro. The efficacy of the plant extracts was tested by antibacterial activity and was used as seed treatment to know the enhancement of seed germination and seedling vigour. Beside promising results given, plant extracts were tried under greenhouse studies to test the efficacy in controlling bacterial blight disease incidence. To get the molecular level of evidence and the estimated defense enzymes in plant extracts, plants were treated, after challenge inoculation, with a target pathogen at different intervals. Adathoda vasica leaf extract significantly reduced the bacterial leaf blight pathogen, X. oryzae pv. oryzae (Xoo) in vitro. Seed treatment was found to be effective in reducing the incidence of the disease under greenhouse condition. Physiological observation of A. vasica treated plants indicated that restriction of pathogen colonization or disease development in plant tissue was correlated with the pronounced increase of peroxidase, PAL, b-1, 3-glucanase, polyphenol oxidase and phenol activity after challenge inoculation with the target pathogen. This investigation clearly demonstrated that the A. vasica leaf extract has the ability to induce the activation of defense enzymes accumulation which can be associated with induction of resistance against rice bacterial leaf blight. Key words: Adathoda vasica, bacterial leaf blight, defense enzymes, induced systemic resistance, rice, Xanthomonas oryzae pv. oryzae.

Rice is a widely consumed staple food for a large part of the world's human population. Approximately 90% of the world's rice is grown in Asian continent and constitutes a staple food for 2.7 billion people worldwide. Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae is one of the devastating diseases of rice. A field experiment was conducted during the year 2016 and 2017 to investigate the influence of different meteorological parameters on BLB development as well as the computation of a predictive model to forecast the disease well ahead of its appearance in the field. The seasonal dataset of disease incidence and environmental factors was used to assess five rice varieties/ cultivars (Basmati-2000, KSK-434, KSK-133, Super Basmati, and IRRI-9). The accumulated effect of two year environmental data; maximum and minimum temperature, relative humidity, wind speed, and rainfall, was studied and correlated with disease incidence. Average temperature (maximum & minimum) showed a negative significant correlation with BLB disease and all other variables; relative humidity, rainfall, and wind speed had a positive correlation with BLB disease development on individual varieties. Stepwise regression analysis was performed to indicate potentially useful predictor variables and to rule out incompetent parameters. Environmental data from the growing seasons of July to October 2016 and 2017 revealed that, with the exception of the lowest temperature, all environmental factors contributed to disease development throughout the cropping season. A disease prediction multiple regression model was developed based on two-year data (Y = 214.3-3.691 Max T-0.508 Min T + 0.767 RH + 2.521 RF + 5.740 WS), which explained 95% variability. This disease prediction model will not only help farmers in early detection and timely management of bacterial leaf blight disease of rice but may also help reduce input costs and improve product quality and quantity. The model will be both farmer and environmentally friendly.

Although Xanthomonas oryzae pv. oryzae (Xoo) has been found to be a bacterial pathogen causing bacterial leaf blight in rice for many years, the molecular mechanisms of the rice-Xoo interaction has not been fully understood. In this study, we found that XanFur of Xoo is a novel ferric uptake regulator (Fur) protein conserved among major pathogenic Xanthomonas species. XanFur is required for the virulence of Xoo in rice, and likely involved in regulating the virulence determinants of Xoo. The expression of xanfur is induced by H2O2, and positively regulated by the global transcriptional regulator Clp. Our results reveal the function and regulation of the novel virulence-related Fur protein XanFur in Xoo, providing new insights into the interaction mechanisms of rice-Xoo.

Bacterial Leaf Blight (BLB) of rice (Xanthomonas oryzae pv. oryzae) is one of the major diseases of rice which can cause 10-95% yield losses. One of the environmentally friendly control efforts is the implementation of a rice cultivation technology package using the Paenibacillus polymyxa as biological agens. The purpose of this study was to determine the effectiveness of the technology package with the use of Paenibacillus polymyxa for controlling BLB disease and increasing production also profit analysis of farming. The research was conducted on 4 ha of rainfed rice fields in Malingping District, Lebak Regency, Banten Province, Indonesia. The environmental design used Nested Block Design with the three treatments:, an environmentally friendly control technology package (Paenibacillus polymyxa), a chemical control technology package (active ingredient methyl thiofanate), and farmers’ existing technology (pesticide mixture). Each treatment consisted of 2 varieties, Ciherang and Inpari 33, with 6 replications. The results showed that the application of environmentally friendly technology packages in the Ciherang variety was able to reduce the intensity of damage by 40.91% greater than the application of pesticides by farmers with an increase in production of 8.31-12.00%. The difference in the profits of farming was 10.52-22.85%.

This study was conducted to assess efficacy of biological control against bacterial leaf blight (BLB) of rice produced by Xanthomonas oryzae pv. oryzae. Five endophytic strains (A1, A2, A3, A13 and A15) and two rhizospherial Bacilli (D29 and H8) were tested for their antagonistic activities against BLB in vitro and in vivo. All seven strains showed high potential of antagonistic activity against X. oryzae pv. oryzae and three phytopathogenic fungi in vitro. Test of 16SrRNA gene sequence were assigned isolates A1, A3 and A13 as Bacillus amyloliquefaciens while isolates A2 and A15 as B. methylotrophicus and B. subtilis, respectively. In greenhouse, four strains of displayed 50.29%–57.86% inhibition rate against the pathogen and significantly increased plant fresh weight from 50.03% to 73.11% and dry weight from 64.11% to 86.65% in treated rice plants. In addition, these strains demonstrated strong capability to produce indole-3-acetic acid, siderophores, solubilizing phosphate and also colonize roots. Real-time quantitative polymerase chain reaction revealed that expression of defense-related genes including OsAOS2, OsJMT1, OsNPR1 and OsPR1b were significantly up-regulated in leaves of D29-exposed rice plants, suggesting that treatment of rice with D29 suppressed BLB through systemic activation of the plant defense system. Therefore, data suggest that Bacillus isolates A13, A15, D29 and H8 support effective antagonistic activity against BLB under greenhouse conditions in addition to their potential to promote growth of rice plants.

Bacillus strains are broadly studied for their beneficial role in plant growth and biological control of plant disease and pest; however, little is known about their underlying mechanisms. In this study, we assessed the controlling and defence‐related mechanisms of three Bacillus strains including rice seed‐associated strain B. subtilis A15, rhizobacterial strains B. amyloliquefaciens D29 and B. methylotrophicus H8, all of which are against bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae. Results indicated that all three strains showed strong biofilm formation ability. The culture filtrates of each strain significantly suppressed the growth and biofilm formation of X. oryzae, while changes in bacterial cell morphology such as cell swell and severe cell wall alterations were observed through the transmission electron microscopy images. PCR analysis revealed that all three strains harbour the antimicrobial‐associated genes that are responsible for biosynthesis of bacillomycin, fengycin, iturin and surfactin. Subsequent real‐time qPCR analysis revealed the upregulated expression of fenD and srfAA genes in D29 and H8, and fenD and ituC genes in A15 during their in vitro interaction with X. oryzae. It suggests that the antibacterial mechanisms of the three strains may be at least partially associated with their ability to secrete corresponding lipopeptides. Interestingly, the applications of the three strains in greenhouse conditions were found to be effective in controlling the BLB disease, which was achieved through the activation of inducing systemic resistance resulted from the enhanced activities of defence‐related enzymes. This is the first report of demonstration of the mode of antibacterial effect of Bacillus strains against X. oryzae. Overall, data from the current study provide valuable information for biological control of BLB disease in rice.

In autumn 2020, leaf blight was observed on rice (Oryza sativa L., variety Zhongzao39, Yongyou9, Yongyou12, Yongyou15, Yongyou18, Yongyou1540, Zhongzheyou8, Jiafengyou2, Xiangliangyou900 and Jiyou351) in the fields of 17 towns in Zhejiang and Jiangxi Provinces, China. The disease incidence was 45%-60%. Initially, water-soaked, linear, light brown lesions emerged in the upper blades of the leaves, and then spread down to leaf margins, which ultimately caused leaf curling and blight during the booting-harvest stage (Fig. S1). The disease symptoms were assumed to be caused by Xanthomonas oryzae pv. oryzae (Xoo), the pathogen of rice bacterial blight. 63 isolates were obtained from the collected diseased leaves as previously described (Hou et al. 2020). All isolates showed circular, smooth-margined, yellow colonies when cultured on peptone sugar agar (PSA) medium for 24h at 28℃. The cells were all gram-negative and rod-shaped with three to six peritrichous flagella; positive for catalase, indole, glucose fermentation and citrate utilization, while negative for oxidase, alkaline, phenylalanine deaminase, urease, and nitrate reductase reactions. 16S rRNA gene sequence analysis from the 6 isolates (FY43, JH31, JH99, TZ20, TZ39 and TZ68) revealed that the amplified fragments shared 98% similarity with Pantoea ananatis type strain LMG 2665T (GenBank JFZU01) (Table S3). To further verify P. ananatis identity of these isolates, fragments of three housekeeping genes including gyrB, leuS and rpoB from the 6 isolates were amplified and sequenced, which showed highest homology to LMG 2665T with a sequence similarity of 95%-100% (Table S3). Primers (Brady et al. 2008) and GenBank accession numbers of gene sequences from the 6 isolates are listed in Table S1 and Table S2. Phylogenetic analysis of gyrB, leuS and rpoB concatenated sequences indicated that the 6 isolates were clustered in a stable branch with P. ananatis (Fig. S2). Based on the above morphological, physiological, biochemical and molecular data, the isolates are identified as P. ananatis. For pathogenicity tests, bacterial suspension at 108 CFU/mL was inoculated into flag leaves of rice (cv. Zhongzao39) at the late booting stage using clipping method. Water was used as a negative control. The clipped leaves displayed water-soaked lesions at 3 to 5 days after inoculation (DAI); then the lesion spread downward and turned light brown. At about 14 DAI, blight was shown with similar symptoms to those samples collected from the rice field of Zhejiang and Jiangxi provinces (Fig. S1). In contrast, the control plants remained healthy and symptomless. The same P. ananatis was re-isolated in the inoculated rice plants, fulfilling Koch's postulates. In the past decade, P. ananatis has been reported to cause grain discoloration in Hangzhou, China (Yan et al. 2010) and induce leaf blight as a companion of Enterobacter asburiae in Sichuan province, China (Xue et al. 2020). Nevertheless, to the best of our knowledge, this is the first report of P. ananatis as the causative agent of rice leaf blight in southeast China. This study raises the alarm that the emerging rice bacterial leaf blight in southeast China might be caused by a new pathogen P. ananatis, instead of Xoo as traditionally assumed. Further, the differences of occurrence, spread and control between two rice bacterial leaf blight diseases caused by P. ananatis and Xoo, respectively need to be determined in the future.

Present investigation was carried out to test the effect of time of application on efficacy of Trichoderma harzianum and Pseudomonas fluorescens formulations against bacterial leaf blight of rice, under field condition. Foliar sprays with bioagent(s) in three time sequences i.e. 7 days before, simultaneously and 7 days after inoculation of Xanthomonas oryzae pv. oryzae inoculation significantly reduced disease severity over check during Kharif 2006 and 2007. Maximum reduction in bacterial leaf blight disease was obtained with the application of T. harzianum on 7 days before inoculation of pathogen during Kharif 2006 and 2007 and maximum increase in grain yield was obtained with the application of PBA-2 seven days before inoculation of pathogen. It was revealed that prophylactic spray of bioagents one week prior to pathogen inoculation was significantly (106 cfu/g) effective in reducing disease severity of bacterial leaf blight of rice as compared to chemical treatment.

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.

Burkholderia glumae and Xanthomonas oryzae pv. oryzae are the primary causes of bacterial disease in rice, capable of reducing production and posing a threat to food availability. This research aims to assess the efficacy of a consortium of five bacteria contained in Mikrobat as bioactivators. The goal is to enhance rice yields, mitigate the severity, and suppress grain rot disease and bacterial leaf blight in rice. The research employed a split-plot design encompassing 12 treatment combinations replicated three times. The experimental setup involved two factors: the main plot consisted of rice varieties-Ciherang, Inpari 4, and Trisakti-while the subplots represented types of bioactivators-Mikrobat, EM4, Trichoderma asperellum, and a control. Results demonstrated Mikrobat’s positive impact compared to the control, with an average of 17.37 productive tillers and higher yields than other bioactivator treatments. Notably, Mikrobat exhibited 50.04% spikelets per panicle and a 1000-grain weight of 19.77 g. The severity of disease with Mikrobat treatment was notably lower than with EM4 and T. asperellum, featuring 4.20% for grain rot disease and 4.41% for bacterial leaf blight. This was significantly different from the control, which showed severity rates of 9.94% for grain rot disease and 7.75% for bacterial leaf blight. Among the three tested varieties, Ciherang demonstrated the best response, displaying higher resistance to both tested diseases. The varietal treatments did not significantly differ from each other. Although Mikrobat’s effects were not significantly distinct from EM4 and T. asperellum, its usage still reduced the severity and suppression of grain rot and leaf blight diseases

The bacterial leaf blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is an economically important and one of the most destructive disease of rice in both irrigated and rainfed environments in Asia. In particular, in recent years, the occurrence of abnormal climate and warming phenomena has produced a good environment for bacterial leaf blight. The rice yield continues to decrease, causing 30 to 50% yield loss. Since bacterial pathogen is difficult to manage by other means effectively, developing host plant resistance is the most effective way to control this disease. So, the study was carried out during rain-fed season (July–November 2019) at Agricultural Research Station (ARS) Gangavati, Karnataka, India to identify the bacterial leaf blight resistance reaction among 22 medium slender genotypes of rice by both phenotyping and genotyping by using molecular markers linked with resistance governing bacterial leaf blight genes. Alongside TN-1 was used as susceptible check and screening was done by artificial clip inoculation method. The disease reactions were recorded one week after inoculation, with Standard Evaluation Scale (SES) for BLB ranging from 0–9, when the susceptible check (TN-1) was completely killed. None of the genotypes showed a resistant reaction, but three genotypes viz. IET-27904, IET-25520 and Rp Bio-226 showed resistant reaction against BLB. The six major BLB resistant genes genetic frequencies varied from 42.85% to 14.28% in the molecular evaluation of promising genotypes for major BLB resistant genes using three Simple Sequence Repeats (SSR) and Sequence Tagged Sites (STS) markers respectively.

HomePlant DiseaseVol. 100, No. 7First Report of a New and Highly Virulent Race of Xanthomonas oryzae pv. oryzae, the Causal Agent of Bacterial Leaf Blight of Rice in Guangxi Province, China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a New and Highly Virulent Race of Xanthomonas oryzae pv. oryzae, the Causal Agent of Bacterial Leaf Blight of Rice in Guangxi Province, ChinaX. L. Chen, Q. Yan, R. F. Li, K. H. Li, and L. J. GaoX. L. ChenSearch for more papers by this author, Q. YanSearch for more papers by this author, R. F. LiSearch for more papers by this author, K. H. LiSearch for more papers by this author, and L. J. GaoSearch for more papers by this authorAffiliationsAuthors and Affiliations X. L. Chen Q. Yan R. F. Li K. H. Li L. J. Gao , Institute of Plant Protection, Guangxi Academy of Agricultural Sciences, Guangxi Key Laboratory of Biology for Crop Diseases and Insect Pests, Nanning, Guangxi, 530007, China. Published Online:13 Apr 2016https://doi.org/10.1094/PDIS-12-15-1400-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae, is one of the most widely distributed and devastating diseases of rice (Oryza sativa) in Asia. BLB can cause yield losses ranging from 20 to 30% and as high as 50% (Mew 1987). Typical BLB symptoms, including tannish gray or white lesions along the veins, were observed in the rice fields in Guangxi, China, in October 2013. Fifteen rice leaves with such symptoms were collected from the infected rice fields. These leaves were cut into approximately 1-cm pieces and homogenized in 9 ml of sterile water by grinding after 1% sodium hypochlorite solution treatment. Diluted homogenates were plated on peptone sucrose agar (PSA) and incubated at 28°C for 3 to 4 days. The nonflat, mucous colonies with yellow, round and smooth margins that developed on the plates were selected for further analysis (Dye et al. 1980). A multiplex polymerase chain reaction assay was performed to distinguish X. oryzae pv. oryzae from other X. oryzae pathovars using genomic DNA of the isolates as template with the primers and cycling conditions as described previously (Lang et al. 2010). Three of the isolates were used to conduct pathogenicity assays on differential rice varieties IR26, Java 14, Nanjing 15, Tetep, and Jingang 30 (Fang et al. 1990). The seeds were sown in plastic trays (50 × 40 × 15 cm) in the greenhouse under a 12-h photocycle at 25 to 28°C, and 30-day-old seedlings were transplanted with row spacing of 25 × 20 cm. Plants were clip-inoculated with a bacterial suspension of 3 × 108 CFU/ml at the booting stage and maintained at 100% humidity for 24 h. Thirty leaves were inoculated with each isolate or with sterilized water (as control). Disease severity was scored 20 days after inoculation by visual assessment of the percentage of the leaf area covered by lesions. Leaves with lesion area <25% and >25% were classified as resistant (R), and susceptible (S), respectively (Fang et al. 1990). The result showed that all the isolates were highly virulent on all the tested rice lines, but no symptoms were observed on water-treated leaves. Colonies recovered on PSA plate from inoculated leaves were identical to field isolates and confirmed by morphology together with PCR, which fulfilled Koch’s postulates. Furthermore, pathogenicity assays using these isolates were performed on several near-isogenic lines (NILs), including IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB13 (xa13), IRBB14 (Xa14), IRBB21 (Xa21), and CBB23 (Xa23). Consequently, all the isolates displayed SRRSRSS on the NILs in contrast to SSSSSRR for the Philippine strain PXO99A. The assays were repeated three times with similar results. Previous studies revealed that CBB23 derived from a cross between a wild rice Oryza rifipogon accession (RBB16) and a susceptible indica rice variety Jingang 30 conferred high resistance to all naturally occurring X. oryzae pv. oryae races tested (Wang et al. 2014). Nevertheless, our research proved that our isolates were highly virulent on many rice lines, including CBB23. To our knowledge, this is the first report of a new and highly virulent race of X. oryzae pv. oryae causing BLB of rice in Guangxi province, China, where it could potentially be destructive under conducive conditions.

Xanthomonas oryzae pv oryzae the Causal Agent of Bacterial Leaf Blight of rice: Isolation, Characterization, and Study of Transposon Mutagenesis. X. oryzae pv. oryzae (Xoo) causes bacterial leaf blight (BLB) of rice (Oryza sativa L.), a major disease that constrains production of the staple crop in many countries of the world. Identification of X. oryzae pv. oryzae (Xoo) was conducted based on the disease symptoms, pathogenicity, morphological, physiological, and genetic characteristics of bacterial cultures isolated from the infected plants. Fifty bacterial isolates predicted as Xoo have been successfully isolated. They are aerobic, rod shaped, and Gram negative bacteria. The isolates were evaluated for their hypersensitivity in tobacco and pathogenicity in rice plant. Fifty isolates induced hypersensitive reaction in tobacco and showed pathogenicity symptom in rice in different length. Based on physiological test, hypersensitivity and pathogenicity reactions, three bacterial isolates strongly predicted as Xoo, i.e. STG21, STG42, and STG46, were non indole formation, non pigment fluorescent, hydrolyzed casein, catalase activity positive, but negative oxidase. Partial sequencing of 16S rRNA genes of STG21 and STG42 showed 80% and 82% homology with X. oryzae, respectively, while STG46 showed 84% homology with X. campestris. Mini-Tn5 transposon mutagenesis of STG21 generated one of the mutants (M5) lossed it’s ability to induce hypersensitive reaction in tobacco plant and deficient in pathogenicity on rice. The lesion length of rice leaf caused by the mutant M5 decreased up to 80%. Keywords: bacterial leaf blight, characterization, isolation, transposon mutagenesis, Xanthomonas oryzae pv. oryzae