Evaluation of Organic Amendments as Eco-Friendly Alternatives for Controlling Rice Bacterial Leaf Blight

1. Introduction

Rice (Oryza sativa L., $2n=2x=24$) is the primary dietary staple for approximately 40% of the global population, with nearly 89% of global production harvested and consumed within Asia. Globally, rice spans an agricultural footprint of 162.06 million hectares, yielding 497.70 million metric tonnes. India holds the largest rice cultivation area in the world (44.16 million hectares) and ranks as the second-largest producer globally, contributing 118.87 million tonnes annually. In the subtropical and temperate agro-climatic zones of Jammu and Kashmir, rice covers approximately 0.27 million hectares, providing vital food security and economic stability.

However, intensive agricultural practices and the widespread cultivation of high-yielding, susceptible cultivars like Basmati-370 have increased the severity of biotic pressures. Among these, Bacterial Leaf Blight (BLB), caused by the biotrophic bacterium Xanthomonas oryzae pv. oryzae (Xoo), is a major limiting factor in rice production. Bacterial leaf blight (BLB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), remains one of the most destructive bacterial diseases affecting rice production worldwide. The disease is prevalent across major rice-growing regions of Asia, Africa, and parts of Latin America, causing yield losses ranging from 10–30% under normal conditions and exceeding 50% during severe epidemics. Recent studies indicate that the emergence of new virulent Xoo races, climate variability, and the widespread cultivation of susceptible cultivars continue to threaten global rice productivity. In India, BLB is a recurring problem in important rice-growing states such as Punjab, Haryana, Uttar Pradesh, Bihar, West Bengal, Karnataka, Andhra Pradesh, and Tamil Nadu. Historically documented in Japan in 1884 and first reported in India (Maharashtra) in 1951, BLB can cause crop yield reductions ranging from 6% to 84% depending on weather conditions, host vulnerability, and crop growth stage.

Recent investigations have revealed considerable genetic and virulence diversity among Xoo populations, particularly in southern India, highlighting the need for continuous pathogen monitoring and deployment of durable resistance genes (Jiang, H. et al. (2026). Current management strategies emphasize resistant cultivars carrying pyramided resistance genes (e.g., Xa4, xa5, xa13, and Xa21), integrated disease management practices, and advanced molecular breeding approaches to enhance sustainable BLB resistance in rice (Cahill, D. ,2025 and Chenhao Li, et al.(2026).

The disease manifests in two distinct phases:

• Leaf Blight Phase: Characterized by water-soaked, linear lesions starting at the leaf tips and moving downward along the margins, creating wavy chlorotic lines that exude amber-colored bacterial drops under high humidity.
• Kresek Phase: A severe systemic vascular wilt that causes systemic leaf curling, gray-green chlorosis, and total plant mortality within 2 to 3 weeks of seedling transplanting. Optimum conditions for disease epidemics include high relative humidity (>70%), frequent monsoonal rainfall, and mild temperatures (22–26°C).

Conventional management strategies rely heavily on chemical bactericides like copper oxychloride and clinical antibiotics such as streptomycin sulphate or oxytetracycline. However, the large-scale spraying of antibiotics in open fields presents serious ecological risks. It drives antibiotic resistance genes in phyllosphere microbial communities, creating a potential pathway for resistance transfer to human pathogens.

Consequently, there is an urgent need to develop cost-effective, environmentally friendly biological control strategies. Plant-derived organic amendments present a viable solution. They contain diverse arrays of bioactive secondary metabolites capable of suppressing plant pathogens while maintaining soil and human health. This study evaluates the in vitro and in vivo efficacy of indigenous organic amendments to establish an efficient alternative framework for BLB management.

2. Materials and Methods

2.1. Experimental Site and Sample Collection

The research was conducted at the Division of Microbiology, Faculty of Basic Sciences, SKUAST-Jammu, Chatha (Latitude: 32.657309° N, Longitude: 74.799092° E). Symptomatic leaf samples exhibiting characteristic wavy, water-soaked lesions were collected from a 40-day-old Basmati-370 crop at the Chatha Research Farm. Collected tissues were transported to the laboratory in sterile polyethylene bags and maintained at 4°C until processed.

2.2. Isolation, Diagnostics, and Pathogen Identification

To confirm the bacterial etiology, infected leaves were subjected to a standard diagnostic ooze test. Excised lesions were cut transversely and submerged in test tubes containing sterile clear water. After 30 to 40 minutes, the emergence of a turbid, yellowish bacterial streaming stream was observed.

For isolation, young symptomatic leaf tissues were cut into 5 mm segments, surface-sterilized with 0.1% sodium hypochlorite (NaOCl) for 30 seconds, and rinsed three times with sterile distilled water. The tissues were transferred to sterile watch glasses containing 5 mL of sterile water for 10 minutes to allow the bacterial cells to diffuse. Loopfuls of the suspension were streaked onto Petri plates containing modified Nutrient Agar (NA) medium (Hi-Media) and incubated in a Biochemical Oxygen Demand (BOD) incubator at 27 ⁰C for 72 to 96 hours.

Pure cultures were isolated from typical single straw-yellow colonies and maintained on NA slants at 4°C for ongoing work. Cellular morphology was assessed using standard Gram staining techniques and evaluated under oil immersion microscopy (100X magnification).

2.3. Preparation of Organic Amendments

Three organic materials—Neem Seed Kernel (NSK), Neem Cake (forming Neem Organic Extract, NOE), and Banana Peel (BP)—were sourced from the Organic Research Farm at SKUAST-Jammu.

• Neem Seed Kernel Extract (NSKE): Dried neem seeds were decorticated using a laboratory blender, and kernels were isolated using an air-driven aspirator. The kernels were milled into a fine powder and sieved.
• Extraction Protocol: For each organic amendment, 100 g of processed raw material was homogenized in 100 mL of sterile distilled water (1:1 w/v). The mixtures were agitated continuously for 1 hour and allowed to steep for 24 hours at room temperature. The crude extracts were filtered through a double-layered muslin cloth followed by Whatman No. 1 filter paper to obtain a 100% standard stock extract.

2.4. In Vitro Antibacterial Screening (Poisoned Food Technique)

The antibacterial activity of the extracts was evaluated using the Poisoned Food Technique. Molten modified Nutrient Agar was cooling-amended with the stock extracts to achieve final volumetric concentrations of 10%, 15%, and 20% (v/v). Aliquots of 100 µL of a fresh Xoo suspension were spread uniformly across the solidified plates using a sterile glass spreader.

Streptomycin sulphate served as the positive chemical control, and unamended NA plates served as the untreated negative control. The plates were arranged in a Completely Randomized Design (CRD) with three replications and incubated at 27 ⁰C . Bacterial colony development was verified at 36, 48, and 72 hours, and qualitatively scored for growth dynamics.

2.5. In Vivo Pot Experiment and Pathogenicity Evaluation

Greenhouse pot experiments were established in a CRD structure featuring four replications to determine the protective capacity of the amendments on Basmati-370 rice plants. The treatments were evaluated at a 10% concentration using a dual-delivery framework: Seed Dip + Foliar Spray.

Seeds were submerged in the respective 10% organic extract formulations before sowing. After transplanting into pots, artificial challenge inoculations were performed using the Pin and Prick Method. Sterile multi-needle arrays were dipped into a 48-hour-old active Xoo broth and used to introduce uniform punctures along the leaf tip margins of healthy plants. Foliar sprays of the corresponding 10% organic treatments were applied systematically post-inoculation.

Disease dynamics were tracked and evaluated using standard epidemiological indices. Percent Disease Incidence (PDI) was calculated according to McKinney (1923):

Percent Disease Severity was calculated using the area-destruction indexing method described by Senevirathna et al. (2016):

2.6. Statistical Analysis

The experimental data were subjected to analysis of variance (ANOVA) techniques appropriate for Completely Randomized Designs. Critical Differences (C.D.), Standard Error of Mean [SE(m)], Standard Error of Difference [SE(d)], and Coefficients of Variation (C.V.) were calculated to determine statistical significance at a 0.05 confidence threshold.

3. Results

3.1. Isolation and Phenotypic Characterization of Xoo

The bacterial pathogen isolated from infected fields showed distinctive morpho-cultural characteristics after 7 days of incubation on modified Nutrient Agar. The colonies were waxy, creamy yellow, circular, smooth, slightly raised, and buttery in consistency. Microscopic examination via Gram staining confirmed that the pathogen consisted of Gram-negative, single or paired short rods, confirming its identity as Xanthomonas oryzae pv. oryzae (Table 1).

3.2. In Vitro Efficacy of Organic Amendments

In vitro screening showed highly variable levels of antibacterial inhibition among the organic inputs (Table 2). At a 10% concentration, Neem Seed Kernel Extract (NSKE) achieved complete inhibition of Xoo, matching the performance of the Streptomycin sulphate positive control. Neem Organic Extract (NOE) provided intermediate control, allowing only slight bacterial growth. In contrast, Banana Peel Extract (BPE) failed to inhibit the pathogen, with bacterial colonies matching the excellent growth observed on the untreated control plates.

Follow-up screenings at higher doses (15% and 20%) verified that NSKE maintained complete suppression of the BLB pathogen, matching the standard chemical antibiotic control (Table 3).

3.3. In Vivo Management in Greenhouse Pot Trials

The plant trials confirmed the in vitro results, showing that organic treatments significantly reduced disease parameters in Basmati-370 rice plants.

3.3.1. Disease Incidence

The application of 10% NSKE as a combined seed dip and foliar spray significantly reduced disease incidence to 16.2%. Plants treated with 10% NOE also showed significant protection, with a disease incidence of 18.8%. BPE was less effective, resulting in an incidence of 23.1%. All organic treatments significantly outperformed the untreated control, which had a disease incidence of 30.9% (Table 4).

3.3.2. Disease Severity

Disease severity followed a similar pattern. The 10% NSKE treatment minimized leaf tissue damage, keeping disease severity to 12.9%. This was followed by 10% NOE at 15.3% and 10% BPE at 19.4%. The untreated control developed severe symptoms, reaching a disease severity of 24.1% (Table 5).

4. Discussion

To sustain global food security, it is essential to increase rice yields through production methods that minimize biotic yield losses. However, intensive reliance on synthetic chemicals has created modern challenges, including environmental pollution and target resistance. This study confirms that plant-derived organic amendments can serve as alternative bactericides to manage Xanthomonas oryzae pv. oryzae (Xoo) morphological profile of our isolated pathogen—characterized by waxy, creamy yellow, mucoid colonies that are Gram-negative short rods—matches classic descriptions of Xoo in literature (Ishiyama, 1922; Ou.S.H, 1985; Swings et al., 1990).

Our in vitro results using the Poisoned Food Technique demonstrated that a 10% concentration of Neem Seed Kernel Extract (NSKE) completely suppresses Xoo replication. This strong antibacterial activity is linked to bioactive tetranortriterpenoids present in neem kernels, such as azadirachtin, nimbin, gedunin, and salannin. These compounds disrupt the structural integrity of the bacterial cell wall, causing lysis and stopping cellular proliferation (Adusei and Azupio, 2022).

These findings align with those of Meena et al. (2004), who reported that neem derivatives produce large zones of inhibition against Xoo at concentrations of 15% and 20%. Similarly, Sawant (2014) demonstrated that NSKE significantly reduces seedling mortality caused by seed-borne bacterial pathogens. The lack of inhibition from Banana Peel Extract indicates that its organic components lack the specific secondary metabolites needed to disrupt Xoo cell walls.

In greenhouse pot trials, the combined application of a 10% NSKE seed dip and foliar spray significantly reduced disease incidence to 16.2% and severity to 12.9%. This confirms its efficacy in live host systems. The seed-dip phase likely neutralizes initial seed-borne inoculum on the seed coat, while subsequent foliar sprays form a protective biochemical barrier over natural leaf openings like hydathodes and stomata, preventing later bacterial entry.

These results are supported by Meena et al. (2010), who found that azadirachtin-based sprays provide up to 59% disease control against leaf blight while improving agronomic yield components. Using these botanical formulations offers a practical approach for integrated disease management, helping to control BLB outbreaks without the ecological risks associated with conventional antibiotics.

5. Summary and Conclusion

Bacterial leaf blight remains a significant threat to global rice production, causing severe crop damage during warm, high-humidity monsoon seasons. In response to the growing risk of antibiotic resistance from agricultural chemical use, this study evaluated organic management options using plant extracts.

The experiments demonstrated that Neem Seed Kernel Extract (NSKE) at a 10% concentration completely inhibits Xoo in vitro. In greenhouse trials, using a 10% NSKE formulation as both a seed dip and foliar spray reduced disease incidence to 16.2% and disease severity to 12.9% in susceptible Basmati-370 rice plants.

Based on these findings, a 10% NSKE treatment protocol can be recommended as an effective, sustainable choice for managing Bacterial Leaf Blight in rice crop. This organic approach offers a dependable alternative to synthetic antibiotics, supporting long-term safety and ecological health in agricultural environments.