Rice is a staple food for millions of people worldwide. In Asia, its importance is even greater, as over 90% of the population relies on rice as a daily dietary essential. In India, rice is of utmost importance as the main cereal crop. It occupies 42% of the nation’s cultivated land. Rice is grown over the largest area in India, covering 43.78 million hectares (FAO, 2023) and globally ranks second in production, with a yield of 118.43 million tonnes, behind China.
       
One of the biggest challenges in rice production is the disease threat caused by harmful fungi, bacteria and viruses. Among these, bacterial leaf blight (BLB) stands out as the most severe disease of rice, causing annual yield losses ranging from 20% to 50% and up to 70% under severe epidemic conditions (Nino-Liu et al., 2006; Mew et al., 1993). This devastating disease is caused by the pathogen Xanthomonas oryzae pv. oryzae and it can drastically reduce yield if not properly managed (Nagaraju et al., 2007; Das et al., 2014).
       
The first recorded outbreak of rice BLB was reported from Japan during 1884, when local farmers spotted its devastating effects (Onasanya et al., 2010). In the present scenario, the disease has become a serious epidemic in many parts of the world, with severe cases leading to crop losses of up to 50%.
       
The Xanthomonas pathogen infects rice plants by entering through hydathodes, stomata, or wounds on the roots and leaves (Nino-Liu et al., 2006). Once inside, it causes leaf wilting and disrupts photosynthesis, leading to yield losses of 20-50% (Adhikari et al., 1995, White and Yang, 2009). Infected plants often produce immature grains, which break more easily during milling, resulting in a higher proportion of broken rice (Khan et al., 2014).

Survey for the assessment of BLB disease
 
A roving survey has been conducted across different zones of Chengalpattu district to collect infected rice leaf samples with bacterial leaf blight. The samples were collected from different rice varieties, including CO 51, Ponni and Gundu. Each sample was labelled with an isolate number, place of collection, variety, geographical coordinates (latitude and longitude) and the PDI was calculated. The collected samples were transported to the laboratory for the successful isolation of the pathogen (Mordue, 1982).
 
Assessment of disease severity and calculation of per cent disease index (PDI)
 
Disease severity of bacterial leaf blight (BLB) was evaluated using the standard evaluation system (SES) for Rice developed by the International Rice Research Institute (2013). The severity of infection was assessed by visually scoring the affected leaves based on the proportion of leaf area showing BLB symptoms (Table 1).


       
The PDI was calculated using the following formula:

 
Isolation of Xanthomonas oryzae pv. oryzae
 
The bacterial leaf blight pathogen was isolated following the maceration technique given by Klement et al., (1990) and Schaad et al., (2001). Freshly infected leaf bits (0.5-1 cm) were excised and surface-sterilized with 1% sodium hypochlorite for 30 seconds. The leaf bits were rinsed three times with sterile distilled water and blotted dry using sterile tissue paper. The leaf bits were transferred into a sterile mortar containing 5-10 mL of sterile distilled water and gently macerated using a sterile pestle. From the prepared dilutions, one mL of the sample was carefully transferred into a sterile petri dish. Each plate was overlaid with about 15-20 mL of molten cooled (45-50°C) nutrient agar (NA), with a gentle swirl. The inoculated plates were incubated at 28±2°C for 48-72 hours.
       
Following incubation, the representative colonies were selected and purified using the streak plate method (Benson, 1990). Quadrant streaking was performed on fresh Nutrient Agar plates to obtain the pure culture and incubated again at 28±2°C for 48-72 hours.
 
Morphological identification of the pathogen
 
The incubated plates were observed for bacterial colonies. The colonies depicting Xanthomonas were sub-cultured. The morphological characters of the study isolates were recorded.
 
Pathogenicity test (Koch’s postulates)
 
To confirm the pathogenic nature of the isolates, Koch’s postulates were carried out under controlled conditions. The bacterial suspension of a single colony was prepared on NA broth and adjusted to approximately 10x-10y  CFU ml^-1. A 48-hour-old virulent culture of all ten isolates was prepared separately and inoculation was accomplished using clip inoculation technique as described by Kauffman et al., (1973). The virulence of each isolate was studied based on symptom expression and severity of the symptom.
 
Molecular characterization of Xanthomonas oryzae pv. oryzae
 
The virulent isolate was identified and subjected to molecular characterization. Genomic DNA was extracted using the modified protocol described by Sambrook et al., (1989). Actively growing bacterial cultures were lysed in extraction buffer containing Tris-HCl, EDTA and SDS, followed by protein precipitation and purification. The quality of the DNA was verified through spectrophotometric analysis and gel electrophoresis.
       
To study the molecular identity, the 16S rRNA gene was amplified using universal bacterial primers27F and 1492R, targeting a region of approximately 1300-1500 bp. PCR amplification was performed in a 25 µL reaction mixture containing genomic DNA, PCR buffer, MgCl‚ dNTPs, primers, Taq polymerase and nuclease-free water. Thermal cycling was performed for 35 cycles with an annealing temperature of 55°C for 30 seconds. The products were gel electrophoresed and resolved in 1% agarose gel.
       
The purified PCR product was subjected to Sanger sequencing at Barcode Biosciences Pvt. Ltd., Bengaluru. The obtained sequence was submitted to GenBank database for retrieving the accession number. BLASTn analysis and phylogeny were performed by retrieving the sequences from the NCBI database using MEGA 12.0 0 (Kumar et al., 2024).

Survey for the assessment of blb disease
 
Disease severity varied considerably across locations and varieties, ranging from 40% to as high as 85% from January 2025 to May 2026 (Table 1). Among the varieties surveyed, the variety CO 51 was found to be severely affected, recording the highest incidence of 85%, suggesting its high susceptibility. In contrast, Ponni and Gundu exhibited moderate levels of infection (40 to 65%). The differences in disease severity observed among the surveyed fields may be due to the varieties cultivated, pathogen density, weather parameters and different agronomical practices.
       
Typical symptoms observed in infected fields included yellowing that began at the leaf tips, water-soaked lesions along the leaf margins and gradual drying of affected tissues. As the disease advances, leaf spots shift from yellow to a pale straw colour. The prominent symptom, viz. wavy, irregular edges on the lesions was observed in all the field (Fig 1). Over time, individual spots merge, creating large blighted patches with those characteristic undulating borders.


       
Similar symptoms, including tip yellowing, water-soaked streaks along leaf margins, irregular lesions with wavy margins and progressive drying, were earlier reported by Ou (1985); Nino-Liu et al. (2006); Kauffman et al. (1973) and Ezuka and Kaku (2000). These observations reinforce the infection process, where the pathogen typically enters through hydathodes or wounds as reported by Swings et al. (1990) and subsequently colonizes the xylem vessels, enabling systemic spread within the plant. The survey revealed that BLB incidence in Chengalpattu district ranged from 40% to 85% in the rice varieties CO 51, Gundu and Ponni, showing the highest incidence of 85% in CO 51, followed by Gundu (45%-65%) and Ponni (40%) (Table 2). These results are consistent with earlier reports by Mew et al., (1993) and Nagaraju et al., (2007), who documented severe BLB outbreaks in susceptible cultivars under favourable conditions. The roving survey conducted in different rice-growing locations of Chengalpattu district revealed considerable variation in bacterial leaf blight severity. The disease was prevalent in all surveyed areas, although the level of infection differed among locations and cultivars. Similar observations were reported by Kanipriya et al., (2024), who identified diverse pathotypes and virulence patterns among Xanthomonas oryzae pv. oryzae isolates collected from different rice-growing regions of Tamil Nadu. The similarity is likely due to comparable agro-climatic factors such as high humidity, warm temperatures and intensive cropping systems that favour rapid pathogen spread.


 
Isolation of Xanthomonas oryzae pv. oryzae
 
The pathogen was successfully isolated on NA media using the maceration technique. All isolates produced uniform colonies that were circular, smooth, slightly raised, bright yellow in colour due to the production of Xanthomonadin pigment, shiny in appearance and distinctly mucoid as a result of extracellular polysaccharide secretion (EPS) (Table 3). These characteristic features confirmed the pathogen’s identity as Xanthomonas oryzae pv. oryzae. The colony characters documented were on par with the reports of Schaad et al., (2001) and Mew et al., (1993), where they observed bright yellow smooth colonies.


 
Morphological identification of the pathogen
 
After an incubation for 48-72 hours the colonies appeared circular with smooth, entire margins and showed a slightly convex to raised profile measuring 1-3 mm in diameter. They displayed a bright, uniform yellow pigmented, shiny colonies with mucoid consistency. The yellow coloration was stable across all isolates, a hallmark of Xanthomonas oryzae pv. oryzae. Growth on NA was moderate to profuse, producing clearly distinguishable colonies with or without spreading or swarming behaviour (Fig 2). The colony traits observed here align closely with the work of Kanipriya et al., (2024), who documented the distinctive yellow, mucoid colonies of Xanthomonas oryzae pv. oryzae from rice fields across Tamil Nadu. Furthermore, Karan et al., (2026) emphasized the importance of colony morphology and cultural characteristics as practical and reliable preliminary criteria for the identification and characterization of plant-associated bacterial pathogens.


       
The colonies were distinctly mucoid in texture, a feature that aligns with earlier descriptions by Schaad et al., (2001) and Mew et al., (1993). The yellow pigmentation is attributed to Xanthomonadin, a pigment known to protect bacterial cells against oxidative stress and ultraviolet radiation, thereby enhancing survival under field conditions. The mucoid consistency reflects EPS secretion, which plays a critical role in virulence by facilitating adhesion, biofilm formation and xylem blockage Denny (1995). The production of EPS is considered an important virulence determinant that enables successful colonization and systemic movement of the pathogen within host tissues. Similar observations on colony morphology and pathogen variability were reported by Kanipriya et al., (2024).
       
Microscopic examination using Gram staining further confirmed the cellular characteristics. Under oil immersion (100X), the bacterial cells-stained pink, indicating their Gram-negative nature.
       
They appeared as short, slender rods (bacilli), measuring approximately 0.5-0.8 µm in width and 1.0-2.0 µm in length. Cells were mostly observed singly, occasionally in pairs and rarely in short chains. The consistent rod-shaped morphology and Gram-negative reaction strongly supported the identification of the isolates as Xanthomonas oryzae pv. oryzae (Fig 3). The morphological characteristics observed in this study correspond well with the taxonomic descriptions of Xanthomonas oryzae pv. oryzae outlined by Bradbury (1986); Schaad et al., (2001). Midha et al., (2017) also reported that diverse isolates of this pathogen consistently exhibit the traits of Gram negative, rod shaped bacteria. Adding to this, Jerish et al., (2022) highlighted the importance of pathogen diversity in bacterial leaf blight and stressed the value of characterizing pathogen populations to improve disease management strategies. Although the isolates examined here shared similar colony morphology, earlier studies have shown that such outward resemblance does not guarantee uniformity in pathogenic potential. In reality, morphologically alike isolates may vary considerably in their virulence and disease causing behaviour (Midha et al., 2017; Jerish et al., 2022; Kanipriya et al., 2024).


 
Pathogenicity test (Koch’s postulates)
 
Pathogenicity assay using clip inoculation method on rice variety CO 51 successfully reproduced the typical BLB symptoms. Inoculation was performed 40 DAS (Days after sowing). Symptom expression was initially observed 10-15 DAI (Days after inoculation). Water-soaked streaks developed near the clipped leaf tips, followed by lesion elongation, yellowing and eventual drying of the infected portions. In contrast, control plants inoculated with sterile distilled water remained completely healthy throughout the observation period.
       
Re-isolation of the bacterium from symptomatic leaves yielded colonies identical to the original isolates, thereby satisfying Koch’s postulates and confirming the causal role of the pathogen (Fig 4).


       
The virulence of ten isolates of Xanthomonas oryzae pv. oryzae was tested under controlled conditions using the rice variety CO 51. The plants produced classic signs of bacterial leaf blight, including water-soaked streaks near the clipped leaf tips, yellowing and gradual drying along the leaf margins. The severity of disease varied noticeably among the isolates. Based on visual observations and the calculated PDI, the isolates were classified into distinct virulence categories (Table 4).


       
Pathogenicity test outcome aligns with Kauffman et al., (1973), who standardized the method for reliable pathogenicity confirmation. The reproduction of identical symptoms and successful re-isolation of the pathogen fulfil Koch’s postulates, validating the causal link. The clip method’s effectiveness lies in its ability to mimic natural infection through hydathodes, ensuring uniform inoculum delivery. A key finding was the variation in virulence among the ten isolates, with Isolate 1 showing the highest PDI. Similar variability has been reported by Shanti et al., (2001) and Wang et al. (1996), who attributed differences to genetic diversity and environmental selection pressures.
 
Molecular characterization of Xanthomonas oryzae pv. oryzae
 
The virulent isolate was subjected to DNA extraction using the lysis buffer method as described by Sambrook et al., (1989). The integrity of the DNA was confirmed through agarose gel electrophoresis before proceeding to amplification. The 16S rRNA gene was targeted using universal bacterial primers 27F and 1492R, which successfully amplified a fragment of approximately 1330 base pairs, producing a single clear band on the gel.
       
The purified PCR product was sequenced and the resulting nucleotide sequence was analysed using BLASTn against the NCBI GenBank database. The sequence exhibited 100% similarity with reported strains of Xanthomonas oryzae pv. oryzae, in NCBI database thereby confirming the molecular identity of the isolate, which is consistent with genomic studies of Xoo strains (Bogdanove et al., 2011). The validated sequence was subsequently deposited in the GenBank under accession number PX945785, providing a permanent molecular reference for future studies.
       
Phylogenetic analysis was performed with Xoo isolates reported worldwide and retrieved from database for analysis. The study isolate was found to be closely clustered with other isolates of Xoo at a bootstrap value of 70% and 1000 replications indicating high nucleotide similarity. Pseudomonas syringae pv. phaseolicola (FJ972539), was used as outgroup. All the isolates were segregated into two major groups. Group I included all the isolates of Xoo whereas Group II had outgroup. Under Group I all the isolates of Xoo diverged into different subgroups. Our study isolate PX945785 was found to be more closely associated with the isolates MZ714131 (Egypt), KX088299 (Andaman and Nicobar), OP071236 (Telangana), MW069710 (Kerala), PX658324 (Korea), PV759764 (Tirunelveli) and MH158537 (China) with 100% nucleotide identity. Interestingly, other isolates of Tamil Nadu including PP165072 (Chidambaram) and OR587908 (Coimbatore) was found to be clustered separately with an identity of 99% (Fig 5).


       
Phylogenetic analysis based on 16S rRNA gene sequences confirmed that the study isolate (PX945785, CGN) from rice, clustered within the Xanthomonas oryzae pv. oryzae (Xoo) clade, forming a well-defined monophyletic group with other reference strains isolated from rice across diverse geographical regions. The tight clustering, supported by short branch lengths (0.02 substitutions per nucleotide position), indicates high sequence similarity and minimal evolutionary divergence among Xoo isolates, suggesting a conserved genetic makeup, as reported by Nino-Liu et al. (2006) and Triplett et al., (2016).
       
Similarly, Midha et al. (2017) have performed phylogenetic analysis for 106 Xoo strains from African, USA and Asia. Their study revealed that Indian Xoo strains, along with a few Asian strains, form a lineage distinct from USA and African strains.
       
Similarly, Koebnik et al., (2021) analysed multiple Xanthomonas oryzae pv. oryzae (Xoo) strains from diverse geographical regions and reported clear lineage differentiation among strains. Their study showed that Asian Xoo strains, including those from India, tend to cluster separately from African strains, indicating geographic structuring and independent evolutionary trajectories within global Xoo populations.
       
The phylogenetic tree clearly distinguished the outgroup, forming a separate branch with greater evolutionary distance, thereby validating the accuracy of tree rooting and highlighting divergence from unrelated taxa. Within the Xoo cluster, the study isolate exhibited close genetic affinity with strains reported from Assam, Egypt, Telangana, Kerala and China, indicating a high degree of genetic conservation across geographically distant populations. Similar patterns of low genetic variability and widespread distribution have been documented by Lee et al., (2005) and Adhikari et al., (1995).
       
Further molecular validation was achieved by amplifying a ~1300-1500 bp 16S rRNA gene fragment, followed by BLAST analysis, which revealed 100% sequence similarity to authenticated Xoo reference strains.
       
Overall, the results provide strong molecular evidence that the isolate PX945785 is correctly identified as Xanthomonas oryzae pv. oryzae. Its close genetic relationship with previously reported strains from different geographical regions highlights the conserved nature of this pathogen. This genetic consistency further supports its role as the causal agent of bacterial leaf blight in rice.

Bacterial Leaf Blight (BLB) remains a major threat to rice cultivation in Chengalpattu district, with the susceptible variety CO 51 showing disease severity as high as 84.44%. Variations in disease severity across locations indicate that environmental conditions, cultivation practices and pathogen diversity strongly influence disease development. Cultural, morphological, pathogenicity and 16S rRNA analyses confirmed that BLB pathogens were closely related to established reference strains, though they differed markedly in virulence. Repeated cultivation of susceptible varieties may be contributing to the emergence of more aggressive pathogen populations. The prevalence of highly virulent isolates underscores the importance of continuous pathogen monitoring, cultivating resistant varieties and adopting integrated disease management practices.

The present study was supported by SRM College of Agricultural Sciences, Baburayanpettai.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.