Endophytic Bacillus velezensis BDKishJoy 6B promotes rice growth and enhances resistance to Magnaporthe oryzae

Abstract

Rice blast, caused by Magnaporthe oryzae, is a persistent threat to rice production in Bangladesh, particularly in the haor (wet-land basin) region, where up to 80% yield loss occurs annually. This study aimed to explore the potential of an endophytic bacterium, Bacillus velezensis BDKishJoy-6B, for promoting growth and enhancing blast tolerance in rice cultivars commonly grown in this vulnerable agro-ecosystem. Fifteen isolates of P. oryzae were collected from haor regions and characterized morphologically and for pathogenicity. The most virulent isolate, KSH01 (GenBank: PQ269871.1), was selected for pathogenicity assays. Antagonistic endophytic bacteria were isolated from rice roots and screened for plant growth-promoting traits. The most effective isolate, BDKishJoy-6B, was identified as B. velezensis (GenBank accession: PQ770697.1). This strain was applied to two rice cultivars (US-2 and BRRI Dhan34) via seed priming, seedling priming, and combined treatments. Growth parameters, yield traits, defense gene expression (OsPOD, OsPAL1, OsPR5), and antioxidant enzyme activities (SOD, POD, APX, PPO, MDA) were assessed under artificial disease pressure. B. velezensis BDKishJoy-6B significantly enhanced rice growth and yield under blast stress, especially in BRRI Dhan34. Notably, seedling priming showed the highest upregulation of defense genes and antioxidant activities, correlating with reduced disease symptoms and increased biomass accumulation. Seedling priming with B. velezensis BDKishJoy-6B offers a promising bio-management strategy for controlling rice blast by modulating host antioxidant and defense pathways, with potential application in haor-based rice production systems.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-29919-x.

Introduction

Rice (Oryza sativa L.) is one of the most important staple crops globally, feeding over three billion people. It serves as a primary source of calories for a significant proportion of the world’s population, with an average per capita consumption of about 57 kg annually¹. Bangladesh ranks as the third-largest producer of rice, contributing significantly to the global rice supply². In Bangladesh, rice is grown across three primary seasons- Aus, Aman, and Boro that covers 11.7 million hectares, accounting for 77% of the total cropped area². Rice plays a critical role in ensuring food security, but its production is severely threatened by various biotic and abiotic factors, particularly plant diseases.

Among the diseases, rice blast caused by Magnaporthe oryzae, also known as Pyricularia oryzae, is considered the most destructive fungal pathogen, causing significant yield losses worldwide. Rice blast can affect all parts of the rice plant, including leaves, nodes, stems, and panicles, often leading to severe reductions in crop yield. This disease is highly prevalent in Bangladesh, especially during the Boro season, where it can result in losses ranging from 28% to as high as 80% in severe cases³.

The haor wetlands in northeastern Bangladesh contribute about 15% of the country’s annual rice production (and roughly 25% of its Boro rice), making them vital for food security but also rendering them especially vulnerable to rice blast disease, which can cause substantial losses in the Boro crop grown there⁴. Rice blast typically begins as small, spindle- or eye-shaped lesions on leaves that have a whitish or grayish center and a reddish-brown to dark brown margin⁵. The disease causes both direct yield losses through poor grain filling and indirect losses through the degradation of plant health and vigor⁶.

Traditionally, rice blast has been managed through the use of chemical fungicides and resistant rice cultivars. Fungicides such as tricyclazole, azoxystrobin, and benomyl have been commonly used to control the spread of rice blast, effectively managing the disease in many cases⁶. However, the reliance on fungicides has raised concerns about environmental pollution, the development of fungicide-resistant strains of M. oryzae, and the impact of chemical residues on human health⁷. The overuse of fungicides not only aggravates these problems but also leads to the degradation of soil quality, increased pesticide resistance, and contamination of water bodies⁸. Furthermore, excessive pesticide use has been linked to poisoning and long-term health risks, particularly in developing countries where pesticide management practices may be inadequate. Given these limitations, there is a growing need to explore sustainable and environmentally friendly alternatives to chemical control.

In response to these challenges, biological control using beneficial microorganisms has emerged as a promising alternative to chemical pesticides. Biological control agents (BCAs), particularly plant growth-promoting bacteria (PGPB), offer a sustainable approach to managing plant diseases, including rice blast. Endophytic bacteria, which reside within plant tissues without causing harm, have been shown to possess significant biocontrol potential. These bacteria suppress pathogen growth through various mechanisms such as competition for nutrients and space, production of antimicrobial metabolites, and induction of systemic resistance in plants⁹. Additionally, endophytic bacteria promote plant growth by enhancing nutrient uptake, producing plant hormones, and improving stress tolerance¹⁰.

Among the many beneficial bacteria, Bacillus velezensis has gained attention for its broad-spectrum biocontrol activity and plant growth-promoting abilities. B. velezensis is a Gram-positive bacterium known for producing a range of antimicrobial compounds, including lipopeptides such as surfactin, fengycin, and iturin, which exhibit strong antifungal and antibacterial properties¹¹. These compounds enable B. velezensis to suppress a variety of phytopathogens, including M. oryzae, the causal agent of rice blast. Moreover, B. velezensis has been shown to enhance plant growth through mechanisms such as nitrogen fixation, phosphate solubilization, and production of phytohormones like indole acetic acid (IAA), which promote root development and overall plant health¹².

Several studies have demonstrated the effectiveness of B. velezensis as a biocontrol agent against M. oryzae. For instance, B. velezensis has been shown to inhibit the growth of M. oryzae both in vitro and in vivo, and its application significantly reduces disease severity and enhances rice yield¹³. The bacterium’s ability to induce systemic resistance in plants further enhances its potential as a biocontrol agent, as it triggers plant defense mechanisms that provide protection against subsequent pathogen attacks¹⁴. The application of B. velezensis in seed treatment or biopriming has been particularly effective in enhancing plant resilience against fungal diseases, suggesting its potential for sustainable Fusarium disease management in maize¹⁵.

Biopriming, the application of beneficial microorganisms to seeds or seedlings before planting, has gained popularity as an effective and sustainable strategy to manage plant diseases and improve crop productivity. In rice, biopriming with B. velezensis has been shown to enhance plant growth, increase antioxidant enzyme activity, and improve the expression of defense-related genes in response to pathogen attack¹⁶. By promoting systemic acquired resistance (SAR), bioprimed rice plants exhibit increased tolerance to fungal infections such as rice blast, resulting in healthier plants and higher yields¹⁷. The potential of B. velezensis in biopriming is attributed to its ability to modulate plant immune responses and improve plant health through the production of antioxidants and other bioactive compounds. Several studies have highlighted the role of B. velezensis in enhancing antioxidant enzyme activity, including peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), which play crucial roles in mitigating oxidative stress during pathogen attack^16,18. By enhancing the plant’s natural defense systems, B. velezensis not only protects against pathogens but also improves overall plant vigor and productivity.

Given the significant challenges posed by rice blast and the promising potential of B. velezensis as a biocontrol agent, this study aims to (i) characterize M. oryzae strains from major haor regions and assess their pathogenicity on rice (ii) identify endophytic bacteria from rice plants in the haor regions, evaluating their antagonistic activity against M. oryzae and (iii) assess the plant growth-promoting traits of selected B. velezensis strains and evaluate the impact of biopriming with B. velezensis on antioxidant enzyme activity and defense-related gene expression in rice. Although the role of B. velezensis as a biocontrol agent has been widely reported, its performance in the unique haor agroecosystem of Bangladesh remains unexplored. This study not only establishes the dual functionality of the endophyte BDKishJoy-6B in both blast suppression and growth promotion but also provides novel evidence of its ability to transcriptionally modulate rice defense and antioxidant pathways. Furthermore, by directly comparing seed, seedling, and foliar priming, we highlight seedling biopriming as the most effective strategy, offering a practical and eco-friendly approach for blast management in haor-based rice systems.

Materials and methods

Experimental sites and period

Field and laboratory experiments were conducted between April and November 2024 at the Department of Plant Pathology, Bangladesh Agricultural University, and the Plant Pathology Division, Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh, Bangladesh. Pot experiments were carried out in the net house of BINA.

Sample collection

Collection of blast-infected plant samples

Neck blast-infected panicles showing characteristic black lesions at the base of the panicle were collected during the Boro season (2023–2024) from 15 haor locations across Brahmanbaria and Kishoreganj districts. Samples were wrapped in sterile paper bags, labeled, and transported to the laboratory for fungal isolation (Supplementary Table 1).

Collection of healthy root samples

Healthy root samples (n = 37) were collected from 37 locations across eight upazilas in the haor region. Roots were washed and stored in sterile zipper bags and preserved at − 20 °C until further analysis 9Supplementary Table 2).

Isolation and characterization of Pyricularia oryzae (anamorph)

Fungal isolation and purification

Blast-infected rachis tissues (3–5 cm) were incubated on moist blotter paper at room temperature (25 ± 2 °C) for 24 h to stimulate sporulation of P. oryzae. Single conidia were transferred to potato sucrose agar (PSA; 20 g sucrose, 200 g potato infusion, 15 g agar per liter) and incubated at 25 °C under alternating 12 h light/dark cycles¹⁹. Pure cultures were maintained on PSA slants at 4 °C.

Morphological characterization

Colony color, surface texture, and radial growth were recorded after 5, 10, and 15 days. Conidial shape, size, and septation were examined under a compound microscope at 400× magnification.

Pathogenicity test

For pathogenicity assessment, 21-day-old seedlings were spray-inoculated with conidial suspension (1 × 10⁵ conidia mL⁻¹) prepared in 0.02% Tween-20. Inoculated plants were covered with black polyethylene sheets for 24 h to ensure high humidity and incubated in the net house. Disease incidence (DI) and percent disease index (PDI) were recorded at 7 days post-inoculation following²⁰.

Molecular identification

Genomic DNA was extracted using the Wizard^® Genomic DNA Purification Kit (Promega, USA). ITS regions were amplified using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′- CTGTTGGTTTCTTTT CCTCCGC-3′) primers²¹. and sequenced. Sequences were aligned using CLUSTALW and phylogenetic trees were constructed in MEGA v11 using the neighbor-joining method with 1000 bootstrap replicates.

Isolation and screening of endophytic bacteria

Isolation and preservation

Surface-sterilized root tissues were macerated in sterile phosphate-buffered saline and serially diluted up to 10⁻⁶. Dilutions were spread on nutrient agar (NA; 5 g peptone, 3 g beef extract, 15 g agar per liter) and incubated at 37 °C for 24 h. Distinct colonies were purified by streaking and preserved in 30% glycerol at − 80 °C.

Antagonistic activity

Dual culture assays were performed on potato dextrose agar (PDA; 20 g dextrose, 200 g potato infusion, 15 g agar per liter). A 5 mm plug of P. oryzae KSH01 was placed at the center of the plate, while a 5 mm bacterial disc (24 h old culture on NA) was placed 2 cm away from the fungal plug. Plates were incubated at 24 ± 2 °C for 7 days²². Radial growth of the fungus was measured every 5 days, and the percent inhibition of radial growth (PIRG) was calculated using Vincent’s formula²³.

Plant growth-promoting traits

Indole-3-acetic acid (IAA) production

Bacterial isolates were inoculated into tryptone broth supplemented with 0.1% L-tryptophan and incubated at 35 °C, 120 rpm, for 48 h. After centrifugation, 2 mL supernatant was mixed with 2 mL Salkowski’s reagent (1 mL 0.5 M FeCl₃ in 50 mL 35% HClO₄). Development of a pink color after 30 min indicated IAA production²⁴.

Siderophore production

Siderophore activity was tested on chrome azurol S (CAS) agar plates²⁵. Development of a yellow-orange halo zone around colonies indicated positive results.

Phosphate solubilization

Isolates were spot-inoculated on Pikovskaya’s agar and incubated at 28 °C for 7 days. Solubilization index was calculated as the ratio of the total halo diameter to colony diameter²⁶. Please look carefully the below equation. Consider the final corrected manuscript.

Ammonia production

Bacterial cultures grown in peptone water (5 mL) were supplemented with 0.5 mL Nessler’s reagent. A brown to yellow coloration indicated ammonia production²⁷.

HCN production

Bacteria were grown on King’s B medium supplemented with 4.4 g L⁻¹ glycine. Whatman filter paper soaked in 0.5% picric acid solution (in 2% Na₂CO₃) was placed inside the lid. A change from yellow to orange-brown indicated HCN production²⁸.

Molecular identification of bacteria

Genomic DNA was extracted, and the 16 S rRNA gene was amplified using universal primers 27 F and 1492R²⁹. PCR products were sequenced, and BLAST analysis was performed against the NCBI database. Phylogenetic analysis was conducted in MEGA v11 using the neighbor-joining method with 1000 bootstrap replicates.

Biopriming and pot trials

Seed biopriming

Surface-sterilized rice seeds were soaked in bacterial suspension (1 × 10⁷ CFU mL⁻¹, prepared in 1% carboxymethyl cellulose) for 12 h at room temperature with gentle shaking³⁰. Treated seeds were air-dried before sowing.

Seedling biopriming

Twenty-one-day-old rice seedlings were immersed in bacterial suspension (1 × 10⁷ CFU mL⁻¹) for 12 h. Immediately after treatment, seedlings were transplanted into pots (20 cm diameter, 4 kg sterilized soil).

Foliar spraying

At 21 days post-transplantation, seedlings were sprayed with bacterial suspension (1 × 10⁷ CFU mL⁻¹) until runoff.

Experimental setup

Pot trials were arranged in a completely randomized design (CRD) with three replications. Treatments included: T₁: Seed priming, T₂: Seedling priming, T₃: Seed + seedling priming, and T₄: Control. Two rice varieties (US-2 and BRRI dhan34) were used. In this study, BRRI dhan34 was considered as popular aman variety for haor areas and US-2 was considered as universal susceptible check variety. Pots were inoculated with P. oryzae @ 1 × 10⁷ CFU/ml at 21 days post-transplantation.

Disease assessment

Disease incidence (DI) and percent disease index (PDI) were recorded at 7-, 14-, and 21-days post-inoculation using standard scales^31,32. The area under disease progress curve (AUDPC) was calculated according to Simko and Piepho (2012)³³.

Enzymatic and molecular responses

Biochemical assays

Leaf samples were collected 48 h post-inoculation for antioxidant enzyme assays:

• Peroxidase (POD): measured following Anushree et al. (2017)³⁴.

• Superoxide dismutase (SOD): assayed according to Kumar et al. (2012)³⁵.

• Polyphenol oxidase (PPO): determined by the method of Mayer et al. (1965)³⁶.

• Ascorbate peroxidase (APX): activity measured following Nakano and Asada (1981)³⁷.

• Malondialdehyde (MDA): content measured as an indicator of lipid peroxidation (Chen & Zhang, 2016)³⁸.

Gene expression analysis

Total RNA was extracted using the SV Total RNA Isolation System (Promega). First-strand cDNA synthesis was performed with the GoScript™ kit. Quantitative PCR was conducted with gene-specific primers for OsPOD, OsPAL1, and OsPR5, normalized against OsActin¹⁶. Reactions were run with SYBR Green in a StepOnePlus Real-Time PCR system (Applied Biosystems). Relative gene expression was calculated using the 2⁻ΔΔCt method.

Statistical analysis

All data were analyzed using R software (v4.3.0). Two-way ANOVA was performed for treatment and cultivar effects, followed by Fisher’s LSD test at p < 0.05. Data are presented as mean ± standard deviation.

Results

Isolation and morphological characterization of Pyricularia oryzae (anamorph)

Fifteen Pyricularia oryzae isolates were obtained from blast-infected rice rachis collected from various haor regions of Bangladesh. After 3–4 days of incubation on moist blotter paper, the fungus sporulated on rachis tissues, producing brown to black structures. Single-spore isolation followed by sequential sub-culturing on potato sucrose agar (PSA) ensured the purity of isolates. Microscopic examination confirmed typical conidial morphology of P. oryzae, consisting of 2–3 celled, pyriform or pear-shaped spores.

Colony morphology

The 15 isolates exhibited significant morphological diversity on PSA medium. Colony colors were grouped into six distinct categories: greyish, greyish black, greyish white, brownish grey, blackish, and whitish. Surface textures were either smooth (7 isolates) or rough (8 isolates), indicating diversity in surface mycelial development. These variations suggest possible adaptive responses or inherent genetic differences among isolates originating from different agroecological zones (Table 1).

Table 1.

Morphological differentiation of Pyricularia oryzae isolated from selected Haor regions.

SL No.	Isolate name	Variety	Location	Surface appearance	Colony color and texture
1	BIJoy01	BRRI dhan-29	Bijoynagar	Smooth	Greyish Black with white cottony mass
2	BIJoy02	BRRI dhan-92	Bijoynagar	Rough	Greyish, light grey center
3	BIJoy03	BRRI dhan-100	Bijoynagar	Smooth	Greyish black
4	KSH01	BRRI dhan-100	Kishoreganj	Rough	Brownish grey
5	NAS01	BRRI dhan-89	Kishoreganj	Rough	Brownish grey with minimum white cottony mass
6	NAS02	BRRI dhan-96	Nasirnagar	Smooth	Greyish black
7	NAS03	BRRI dhan-81	Nasirnagar	Smooth	Black and grey
8	SARL01	BRRI dhan-29	Nasirnagar	Rough	Greyish with white cottony mass
9	SARL02	BRRI dhan-100	Sarail	Rough	Whitish grey
10	NIK01	BRRI dhan-92	Sarail	Smooth	White
11	NIK02	BRRI dhan-96	Nikli	Smooth	Greyish brown
12	TARL01	BRRI dhan-89	Nikli	Rough	Grey and white
13	TARL02	BRRI dhan-100	Tarail	Rough	Whitish grey with concentric rings
14	KSH02	BRRI dhan-100	Tarail	Smooth	Greyish black with concentric ring
15	KOTY01	BRRI dhan-29	Kotiyadi	Rough	Brownish grey

Conidial morphology

Conidial characteristics were highly variable across isolates. Based on shape, four morphological types were observed: fusoid, fusoid with tapering ends, ventricose, and ovoid. Spore sizes also varied among isolates, forming five size groups. KSH01, NAS03, and NIK02 exhibited the largest conidia ((23.44–26.80) × (8.08–8.5) µm), while TARL01 and NIK01 showed the smallest. Such variability in conidial morphology and size is often associated with pathogenic fitness and environmental adaptability (Table 2).

Table 2.

Morphological variation of Pyricularia oryzae.

Shape of conidia	Isolate name	Conidial size
		Length (µm)	Breadth (µm)
Fusoid	BIJoy02, BIJoy03, TARL02	20.54–20.86	7.40–7.56
Fusoid with long tapering end	TARL01, KSH02, NIK01	20.12–23.01	6.30–6.52
Ventricose	SARL01, KOTY01, SARL02	26.27–27.55	6.65–6.90
	KSH01, NIK02, NAS03	23.44–26.80	8.08–8.5
Ovoid	BIJoy01, NAS01, NAS02	18.89–20.25	8.01–8.9

Radial growth

All isolates were grown on PSA for 15 days, and radial mycelial growth was measured at 5-day intervals. KSH01 exhibited the fastest growth rate (7.9 cm at 15 DAI), whereas TARL01 was the slowest (3.59 cm). These differences in radial expansion reflect variability in growth kinetics, potentially linked to virulence (Table 3).

Table 3.

Morphological differentiation of Pyricularia oryzae based on colony growth on culture media at different days after inoculation (DAI).

Isolate name	Radial mycelial growth (cm)
	5 DAI	10 DAI	15 DAI
BIJoy01	3.9 c	6.9 a	7.3 cd
BIJoy02	2.5 h	3.67 h	4.74 h
BIJoy03	3.28 d	4.8 f	5.9 f
KSH01	4.55 a	6.95 a	7.9 a
NAS01	3.15 e	5.4 e	6.48 e
NAS02	2.87 g	3.55 h	4.71 h
NAS03	3.95 c	6.6 b	7.5 bc
SARL01	2.9 fg	3.94 g	5.3 g
SARL02	2.14 i	3.57 h	4.65 h
NIK01	1.5 j	2.95 j	3.75 i
NIK02	4.5 a	6.7 b	7.6 b
TARL01	1.25 k	2.8 j	3.59 j
TARL02	2.2 i	3.25 i	4.03 i
KSH02	4.05 b	6.1 c	7.1 d
KOTY01	2.98 f	5.83 d	7.45 bc
LSD	0.05	0.05	0.05
SE	0.003	2.05	0.048
CV (%)	1.83	0.01	2.48

Significant values are in bold.

Pathogenicity assessment of P. oryzae isolates

The virulence potential of the 15 isolates was evaluated on the susceptible rice cultivar under net-house conditions. Small lesions appeared by 3–5 DAI and gradually developed into spindle-shaped lesions with grey centers. Disease incidence (DI) varied from 10% to 70%, while percent disease index (PDI) ranged from 6% to 48% (Table 4).

Table 4.

Pathogenicity of the isolates based on disease incidence (DI) and percent disease index (PDI) at 7 days after inoculation (DAI).

Sl. No	Isolate name	DI at 7DAI	PDI at 7DAI
1	BIJoy01	23.33 ± 3.33 de	20.67 ± 1.77 de
2	BIJoy02	16.67 ± 3.33 ef	14.00 ± 1.15 f
3	BIJoy03	33.33 ± 3.32 cd	26.67 ± 1.77 c
4	KSH01	70.00 ± 3.33 a	48.00 ± 3.05 a
5	NAS01	40.00 ± 5.77 bc	28.00 ± 1.15 c
6	NAS02	20.00 ± 5.77 ef	15.33 ± 1.77 ef
7	NAS03	33.33 ± 3.33 cd	23.33 ± 2.90 cd
8	SARL01	16.67 ± 3.33 ef	14.67 ± 0.66 f
9	SARL02	16.67 ± 3.33 ef	14.67 ± 2.40 f
10	NIK01	10.00 ± 0.00 f	28.00 ± 0.66 c
11	NIK02	36.67 ± 3.33 c	28.00 ± 2.31 c
12	TARL01	13.33 ± 3.33 ef	6.00 ± 1.15 g
13	TARL02	20.00 ± 5.77 ef	10.67 ± 1.77 fg
14	KSH02	33.33 ± 3.33 cd	23.33 ± 2.40 cd
15	KOTY01	50.00 ± 5.77 b	34.67 ± 2.40 b
CV (%)		25.27	16.27
SE		17.76	3.85
LS		0.05	0.05

Significant values are in bold.

Isolate KSH01 showed the highest pathogenicity, with DI and PDI of 70% and 48%, respectively. On the contrary, TARL01 and NIK01 were the least virulent. Given its high virulence and rapid growth, KSH01 was selected for molecular identification and in vitro antagonism assays.

Molecular identification and phylogenetic positioning of KSH01

The internal transcribed spacer (ITS) region of KSH01 amplified a 545 bp product. BLAST analysis confirmed 99% identity with multiple P. oryzae isolates (Table 5). Phylogenetic tree construction using the neighbor-joining method placed KSH01 in a distinct clade closely related to POIIRR 4, suggesting geographic or functional divergence (Fig. 1).

Table 5.

Closest relatives of Pyricularia oryzae KSH01 isolate.

Isolate	Closest relatives	Accession no.	Alignment	Homology (%)
KSH01 (PQ269871.1) (545 bp)	Pyricularia oryzae isolate PLK010901	OR492535.1	516/520	99
	Pyricularia oryzae isolate IC1	OQ552821.1	516/520	99
	Pyricularia oryzae isolate MoK19-48	MT757310.1	513/517	99
	Pyricularia oryzae isolate MoK19-20	MT757291.1	513/517	99
	Pyricularia oryzae isolate SSK010103	OR492534.1	515/520	99
	Pyricularia oryzae isolate POIIRR 4	ON409978.1	517/522	99

Fig. 1.

Neighbor-joining phylogenetic tree showing the relationships between Pyricularia oryzae KSH01 and its closely related strains.

Isolation and antagonistic activity of endophytic bacteria

From 46 endophytic bacterial isolates derived from rice root tissues, dual culture assays were performed to assess antifungal potential against KSH01 (Table 6). Eleven isolates demonstrated > 89.75% inhibition of fungal growth at 15 DAI (Fig. 2). Among them, BDKishJoy-6B achieved the highest inhibition (93.75%), followed by BDBrCham-7 A (93.13%) and BDBrBij-3B (92.29%) (Fig. 3). Radial growth of KSH01 was significantly suppressed by these isolates, indicating strong mycostatic activity.

Table 6.

Effect of different endophytic bacterial isolates on in vitro suppression of Pyricularia oryzae KSH01 responsible for blast disease of rice.

Sl No.	Isolate	Mycelial Growth of KSH01			% Inhibition over control
		5DAI	10DAI	15DAI
1	Control	3.90 a	5.10 a	8.00 a	0.00 s
2	BDBrNas-1 A	0.60 qr	0.80 a	0.80 q-s	90.00 a-c
3	BDBrNas-1B	0.80 p-r	1.43 b	1.82 p	77.29 d
4	BDBrNas-1 C	2.05 ij	2.90 bc	4.00 j	50.00 j
5	BDBrSar-2 A	2.10 i	2.47 cd	3.00 m	62.50 g
6	BDBrSar-2B	3.10 c-g	3.70 cd	4.45 i	44.38 k
7	BDBrSar-2 C	2.90 d-h	4.60 de	5.43 fg	32.08 mn
8	BDBrSar-2 C	3.02 d-g	4.28 d-f	5.00 h	37.50 l
9	BDBrBij-3 A	2.73 gh	3.48 d-f	6.02 de	24.79 op
10	BDBrBij-3B	0.50 r	0.50 e-g	0.62 s	92.29 a
11	BDBrBij-3 C	0.65 qr	0.71 fg	0.79 q-s	90.04 a-c
12	BDBrBij-3D	0.90 pq	2.60 gh	2.92 m	63.54 g
13	BDBrBij-3E	1.69 j-l	2.59 s-v	4.99 h	37.58 l
14	BDBrBij-3 F	1.30 m-o	3.11 tuv	3.78 jk	52.71 ij
15	BDKishTar-4 A	0.53 qr	0.55 gh	0.57 s	92.88 a
16	BDKishTar-4B	0.60 qr	0.77 gh	0.82 q-s	89.75 a-c
17	BDKishTar-4 C	1.55 lm	2.05 hi	2.85 mn	64.38 fg
18	BDKishTar-4D	0.90 pq	1.02 v	1.08 qr	86.50 bc
19	BDKishTar-4E	3.17 b-e	4.60 ij	6.59 bc	17.67 qr
20	BDKishNik-5 A	0.50 r	0.60 i-k	0.67 rs	91.67 ab
21	BDKishNik-5B	3.27 b-d	3.85 j-l	5.73 ef	28.33 no
22	BDKishNik-5 C	4.02 a	6.02 j-l	7.02 b	12.29 r
23	BDKishNik-5D	3.13 b-f	4.30 j-m	5.17 gh	35.42 lm
24	BDKishNik-5E	1.62 k-m	3.03 j-l	4.09 ij	48.83 ij
25	BDKishJoy-6 A	0.60 qr	0.67 k-n	0.78 q-s	90.21 a-c
26	BDKishJoy-6B	0.50 r	0.50 l-n	0.50 s	93.75 a
27	BDKishJoy-6 C	2.52 h	4.45 mn	5.10 gh	36.25 lm
28	BDKishJoy-6D	0.92 o-q	1.68 mn	2.18 op	72.75 de
29	BDKishJoy-6E	0.80 p-r	0.80 t-v	1.05 qr	86.88 bc
30	BDKishJoy-6 F	0.80 p-r	1.03 uv	1.20 q	84.96 c
31	BDKishJoy-6G	1.76 i-l	3.05 v	3.82 jk	52.20 ij
32	BDBrCham-7 A	0.50 r	0.50 mn	0.55 s	93.13 a
33	BDBrCham-7B	3.15 b-e	5.88 no	6.19 cd	22.58 pq
34	BDBrCham-7 C	2.85 e-h	4.02 op	4.97 h	37.92 l
35	BDBrCham-7D	1.07 op	2.63 v	3.03 m	62.08 g
36	BDBrCham-7E	2.03 ij	2.50 v	3.13 lm	60.92 gh
37	BDKishIcha-8 A	0.55 qr	0.73 p	0.79 q-s	90.04 a-c
38	BDKishIcha-8B	3.40 bc	4.07 pq	4.19 ij	47.63 jk
39	BDKishIcha-8 C	0.82 p-r	1.14 qr	2.04 p	74.50 d
40	BDBrBuri-9 A	3.50 b	4.97 qr	5.82 d-f	27.29 n-p
41	BDBrBuri-9B	1.13 n-p	1.38 rs	2.48 no	69.00 ef
42	BDBrBuri-9 C	1.52 l-n	2.70 r-t	3.53 kl	55.83 hi
43	BDBrAru-10 A	2.76 f-h	3.68 r-u	5.14 gh	35.79 lm
44	BDBrAru-10B	1.97 i-k	2.03 s-v	3.98 j	50.21 j
45	BDBrAru-10 C	0.50 r	0.55 s-v	0.72 rs	91.04 ab
46	BDBrAru-10D	2.52 h	3.17 ij	3.76 jk	53.00 ij
	SE	0.16	0.24	1.04	3.87
	CV (%)	0.63	0.64	1.65	0.44

Fig. 2.

In vitro suppression of P. oryzae KSH01 by 9 endophytic representative bacterial isolates which are statistically similar (% Inhibition 89.75–93.75).

Fig. 3.

In vitro suppression of Pyricularia oryzae KSH01 by representative endophytic bacterial isolates B. velezensis BDKishJoy-6B (% Inhibition is 93.75) (right side) compare to control (left side).

Functional screening for plant growth promoting traits

The top 11 antagonistic isolates were subjected to a series of biochemical assays for plant growth promotion:

Indole-3-Acetic acid (IAA): Four isolates tested positive for IAA production, notably BDKishJoy-6B, which produced a pronounced cherry-red layer (Table 7; Fig. 4). IAA production is essential for root development and stress tolerance.

Table 7.

Response of different antagonistic endophytic bacterial isolates to plant growth promoting activities.

Sl No.	Isolate	Different plant growth promoting activities
		IAA production	Siderophore production	Phosphate solubilization	Ammonia production	HCN production
1	BDBrBij-3 C	−	+	−	−	+
2	BDKishTar-4B	+	−	+	+	+
3	BDBrAru-10 C	−	+	−	+	−
4	BDKishJoy-6B	+	+	+	+	+
5	BDBrCham-7 A	−	−	+	+	−
6	BDKishIcha-8 A	−	+	−	+	+
7	BDBrBij-3B	+	−	−	+	+
8	BDKishNik-5 A	−	+	+	+	−
9	BDKishJoy-6 A	−	+	+	+	+
10	BDKishTar-4 A	+	+	+	+	+
11	BDBrNas-1 A	−	+	−	+	+

“+’’ indicates positive response to test & “−” indicates negative response to test.

Fig. 4.

Positive reaction of BDKishJoy-6B to the plant growth promoting determinants compared with control (A = HCN production, B = Siderophore production, C = Phosphate solubilization assay, D = Ammonia production & E = IAA positive).

Siderophore production: Eight isolates exhibited yellow halo zones on CAS agar, with BDKishJoy-6B producing the largest zone (2.46 cm) (Table 7, Nine and Fig. 4). Siderophore production helps in iron chelation and limits pathogen access to this essential micronutrient.

Phosphate solubilization: Six isolates solubilized phosphate on Pikovskaya’s agar. BDKishJoy-6B had the highest solubilization index (4.04), further enhancing its utility in promoting nutrient availability (Tables 7 and 8; Fig. 4).

Table 8.

Phosphate solubilization efficiency determined by phosphate solubilization index (PSI).

Isolate ID	Diameter of Halo zone (cm)	Phosphate Solubilization Index (PSI)
BDKishTar-4 A	1.25 ± 0.01 c	2.46 ± 0.03 c
BDKishJoy-6 A	2.25 ± 0.03 b	3.68 ± 0.07 b
BDKishTar-4B	1.49 ± 0.03 f	1.75 ± 0.03 e
BDBrCham-7 A	0.95 ± 0.05 e	1.72 ± 0.02 e
BDKishJoy-6B	2.47 ± 0.02 a	4.04 ± 0.05 a
BDKishNik-5 A	1.08 ± 0.02 d	2.03 ± 0.04 d
CV (%)	3.19	2.85
SE	0.003	0.0055

Ammonia and HCN production: Ten isolates produced ammonia, and eight produced HCN, both of which contribute to nutrient cycling and pathogen suppression. BDKishJoy-6B tested positive for both traits (Table 7; Fig. 4).

Based on overall performance, BDKishJoy-6B was selected for further net-house and molecular assays.

Safety and molecular characterization of BDKishJoy-6B

The hypersensitivity test on tobacco leaves confirmed that BDKishJoy-6B is non-pathogenic to non-host plants (Supplementary Fig. 1). Molecular identification via 16 S rRNA sequencing revealed 99% similarity with Bacillus velezensis (Accession PQ770697.1). Phylogenetic analysis grouped it closely with other B. velezensis strains, validating its identity and biosafety (Fig. 5).

Fig. 5.

Neighbor-joining phylogenetic tree showing the relationships between BDKishJoy-6B and its closely related strains.

Gene expression of defense-related markers

Quantitative RT-PCR analysis evaluated three defense-related genes (OsPOD, OsPAL1, OsPR5) in BRRI dhan34 and US-2 cultivars under treatments T₁ (seed biopriming), T₂ (seedling biopriming), and T₃ (foliar spraying) (Fig. 6). BRRI dhan34 under T₂ showed the highest upregulation: OsPOD (69.95-fold), OsPAL1 (25.33-fold), and OsPR5 (239.96-fold). In US-2, gene expression was comparatively lower or suppressed. This suggests that BDKishJoy-6B enhances plant immunity through transcriptional modulation of defense genes (Table 9).

Fig. 6.

The antifungal substances of BDKishJoy-6B induced defense related gene expression in rice plants against Pyricularia oryzae KSH01.

Table 9.

Siderophore production efficiency determined by diameter of yellow zone (cm) in CAS agar plate.

Isolate ID	Diameter of halo zone (cm)
BDKishNik-5 A	1.61 ± 0.05 e
BDKishTar-4 A	2.02 ± 0.05 c
BDBrAru-10 C	1.67 ± 0.02 e
BDBrNas-1 A	1.68 ± 0.01 e
BDBrBij-3 C	2.35 ± 0.03 ab
BDKishJoy-6 A	2.25 ± 0.08 b
BDKishTar-4B	1.83 ± 0.02 d
BDKishJoy-6B	2.46 ± 0.04 a
CV (%)	3.53
SE	0.005

Antioxidant enzyme activity

BDKishJoy-6B application significantly increased antioxidant enzyme activity in both cultivars. POD activity reached 1123.56 U/min/g FW in BRRI dhan34 (T₂). Similarly, SOD (4.87 U/mg protein), APX (1.26 µmol/min/mg protein), and PPO (1.26 ΔAbs/min/g FW) were highest in T₂-treated BRRI dhan34. In contrast, MDA levels, indicative of lipid peroxidation, were lowest in T₂ (3.66 nmol/g FW), confirming reduced oxidative stress (Fig. 7).

Fig. 7.

Effect of Bacillus velezensis BDKishJoy-6B on the antioxidant enzyme activity of rice plants against Pyricularia oryzae KSH01.

Disease suppression and biocontrol efficacy in pot experiments

Disease incidence (DI), percent disease index (PDI), and area under disease progress curve (AUDPC) were significantly reduced under BDKishJoy-6B treatments (Figs. 8 and 9). At 21 DAI, BRRI dhan34 treated with T₂ showed the lowest DI (23.33%) and PDI (19.33%), with an AUDPC of 406. In contrast, untreated control (T₄) exhibited 86.67% DI and 76% PDI with an AUDPC of 1596. Biocontrol efficacy reached a peak of 74.85% under T₂ treatment.

Fig. 8.

Effect of BDKishJoy-6B on the suppression of disease incidence of rice blast disease in pot experiment at 7, 14 and 21 days after inoculation (DAI), Here, T₁ = Seed biopriming with Bacillus velezensis BDKishJoy-6B, T₂ = Seedling biopriming with Bacillus velezensis BDKishJoy-6B, T₃ = Foliar spraying with Bacillus velezensis BDKishJoy-6B, T₄ = Control, V₁ = US-2 (susceptible check), V₂ = BRRI Dhan34.

Fig. 9.

Percent disease index (PDI) and area under disease progress curve (AUDPC) of two rice cultivars treated with Bacillus velezensis BDKishJoy-6B with four treatments against Pyricularia oryzae KSH01.

Enhancement of agronomic traits

Yield-contributing parameters were positively influenced by BDKishJoy-6B. In BRRI dhan34, T₂ treatment resulted in the highest 1000-grain weight (15.13 g), shoot length (147 cm), root length (39.44 cm), and root dry weight (58.79 g). Similar improvements were noted in US-2, though to a lesser extent (Table 10). These enhancements indicate that BDKishJoy-6B promotes growth and yield through multiple synergistic mechanisms.

Table 10.

Effect of BDKishJoy-6B on the yield parameter of two rice cultivars in pot experiment.

Cultivar	Treatments	1000 grain weight (g)	Shoot length (cm)	Root length (cm)	Root dry weight (g)
US-2	T1	16.37 ± 0.87 b	109.56 ± 7.62 d	27.33 ± 2.87 bc	57.11 ± 3.59 ab
	T2	19.37 ± 0.83 a	116.22 ± 5.17 c	32.00 ± 5.32 b	39.74 ± 2.84 cd
	T3	13.6 ± 0.83 c	116.89 ± 9.41 c	25.11 ± 2.52 cd	36.32 ± 3.91 cd
	T4	13.03 ± 0.57 c	104.78 ± 3.8 d	20.33 ± 3.04 d	34.99 ± 1.85 d
BRRI dhan34	T1	11.87 ± 0.47 d	145.67 ± 7.62 ab	30.44 ± 0.882 b	52.7 ± 3.06 b
	T2	15.13 ± 0.25 b	147 ± 5.57 a	39.44 ± 2.96 a	58.79 ± 3.58 a
	T3	10.1 ± 0.52 e	140.22 ± 6.3 b	27.33 ± 4.92 bc	55.89 ± 3.78 ab
	T4	9.6 ± 0.53 e	139.67 ± 5 b	25.22 ± 1.99 cd	41.8 ± 3.62 c
CV (%)		4.89	5.08	11.8	7.27
SE		0.15	4.67	1.25	3.72
LS		0.05	0.05	0.05	0.05

Here, T₁ = Seed biopriming with Bacillus velezensis BDKishJoy-6B, T₂ = Seedling biopriming with Bacillus velezensis BDKishJoy-6B, T₃ = Foliar spraying with Bacillus velezensis BDKishJoy-6B, T₄ = Control, CV = Coefficient of variation, SE = Standard error, LS = Level of significance.

Discussion

The production of rice is severely challenged by fungal pathogens, among which Magnaporthe oryzae (syn. Pyricularia oryzae), the causal agent of rice blast, is the most devastating. In Bangladesh, particularly in the Haor agroecosystem, yield losses can reach up to 80% during the Boro season³⁹. Given the limitations of chemical control, biological approaches, particularly using endophytic bacteria, have emerged as promising alternatives. This study explored the potential of endophytic Bacillus velezensis BDKishJoy-6B as a dual-function bioagent—both as a plant growth promoter and as a modulator of defense mechanisms in rice against M. oryzae.

Fifteen P. oryzae isolates collected from the Haor region displayed marked cultural and morphological diversity. Variability in colony pigmentation, radial growth, and spore characteristics was consistent with earlier reports⁴⁰. The isolate KSH01 showed the highest growth rate and pathogenicity, confirming its virulence and suitability for downstream biocontrol assays. Such diversity reflects the adaptive versatility of M. oryzae under the waterlogged Haor ecosystem, underscoring the need for broad-spectrum, sustainable management strategies. In the present study, ITS sequence analysis was used to confirm the identity of the most virulent isolate, KSH01, as Pyricularia oryzae. While ITS is a well-established fungal barcode marker, it offers limited resolution at intra-species level. Future studies should therefore employ multi-locus sequence typing (MLST) using additional housekeeping genes such as TEF1-α, β-tubulin, and calmodulin to better resolve genetic diversity and evolutionary relationships among P. oryzae populations.

Among 46 bacterial isolates screened, BDKishJoy-6B emerged as the most effective antagonist, inhibiting > 93% of P. oryzae KSH01 in dual culture. This aligns with previous reports that B. velezensis produces antifungal metabolites such as surfactins, iturins, and fengycins, which disrupt fungal membranes and interfere with appressorium formation^41,42. In addition, BDKishJoy-6B expressed multiple plant growth-promoting traits (IAA, siderophore, phosphate solubilization, ammonia, and HCN production). These attributes enhance nutrient availability and indirectly strengthen host resistance^43,44. Its particularly high phosphate solubilization and siderophore production are notable, given the nutrient-deficient, flood-prone soils of the Haor region^45,46.

Seedling biopriming with BDKishJoy-6B activated key defense genes (OsPOD, OsPAL1, and OsPR5), all central to the salicylic acid-mediated systemic acquired resistance pathway (Park et al., 2012). The striking 239-fold upregulation of OsPR5 in T₂ treatment mirrors the responses reported for other B. velezensis strains¹⁶. Upregulation of OsPOD and OsPAL1 further demonstrates enhanced ROS detoxification and lignification processes^47–49. These results confirm that B. velezensis can transcriptionally prime rice immunity.

Antioxidant enzyme activity was also strongly enhanced. Significant increases in SOD, POD, PPO, and APX levels, coupled with reduced MDA content, point to an improved redox balance in treated plants. Such modulation of oxidative stress responses is consistent with previous findings^50–52. This suggests that BDKishJoy-6B provides dual protection: activating systemic resistance while mitigating oxidative damage during pathogen challenge.

Integration of molecular, physiological, and phenotypic data confirmed that BDKishJoy-6B substantially suppressed blast disease. Seedling biopriming reduced incidence to 6.67% and achieved a biocontrol efficacy of 74.85%, outperforming seed priming and foliar spray. This agrees with earlier studies showing superior disease suppression through root colonization and early endophyte establishment^16,53,54. Colonization at the seedling stage may also prevent early root infection by M. oryzae⁵⁵.

Importantly, BDKishJoy-6B not only restricted disease but also improved agronomic performance, with significant gains in 1000-grain weight, shoot and root length, and root biomass. This dual benefit parallels findings with other Bacillus strains^56,57. Its capacity to simultaneously promote growth and suppress disease positions BDKishJoy-6B as a valuable component of climate-resilient rice production, particularly in vulnerable Haor ecosystems. However, while pot experiments are promising, further field-scale validation remains essential.

Conclusion

This study demonstrated that endophytic Bacillus velezensis BDKishJoy-6B exhibits a dual role in rice by enhancing plant growth and conferring tolerance against Magnaporthe oryzae through the activation of defense-related and antioxidant genes. The strain not only promoted morphological and physiological improvements but also triggered molecular responses associated with induced systemic resistance. These findings highlight the potential of BDKishJoy-6B as a sustainable alternative to chemical inputs for managing rice blast and improving crop productivity especially in the haor region of Bangladesh. Further large-scale field evaluations under diverse agroecological conditions are essential to validate its efficacy and stability. Additionally, formulation development, compatibility with existing crop management practices, and multi-pathogen bioefficacy testing should be prioritized to facilitate its commercialization as a broad-spectrum bioinoculant.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

S.A. - Material preparation, data collection and analysis, First draft of Manuscript ; S.C. , M.M.H. and T.R.- Material preparation, data collection and analysis, manuscript review M.A.R.K.- Manuscript review and making final draft of manuscript, supervision.

Funding

This research was supported by The City Bank Limited, Bangladesh under the project number: 2023/14/Other.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.