This study was carried out at ICAR-National Institute of Biotic Stress Management (NIBSM), Raipur, Chhattisgarh, India. BLB-infected samples were collected from five states of India, viz., Chhattisgarh (Raipur 21.24 N 81.633 E), Odisha (Cuttack 20.46 N 85.8792 E), Meghalaya (Umiam 25.676 N 91.927 E), Tripura (Agartala 23.831 N 91.287 E), and Assam (Guwahati 26.7269 N 94.2038 E), whereas rice field samples for bacteriophage isolation were collected from all districts of Chhattisgarh (Latitude 17°46′ N to 24°5′ North and Longitude 80°15′ E to 84°20′ East) and adjoining states. Further details of sampling locations of isolated phages are mentioned in Table 1.

Diseased rice leaf samples showing typical bacterial leaf blight symptoms were collected from different rice fields during Kharif 2017 from different agroclimatic zones as mentioned earlier in the studied area. Samples were collected based on the random sampling method following standard sampling protocol. The leaf samples were washed with tap water, surface-sterilized with 1% sodium hypochlorite for 30 s, then washed in sterilized distilled water, and air-dried. Surface-sterilized leaf tissues were dried on sterile filter paper and cut into small pieces of approximately 5 mm × 5 mm in size (Bradbury, 1970). Six to seven tissue pieces were placed on peptone sucrose agar (PSA) plates (peptone 10 g; L-glutamic acid 1 g; sucrose 10 g; and agar 15 g per liter) and incubated at 28°C for 2–7 days. Yellow round mucoid colonies convex appearing with smooth margins on the Petri plates were selected and purified three times on PSA as a single colony. Isolates were subjected to Gram staining, potassium hydroxide test (3% KOH; Sulsow et al., 1982), oxidase test, and catalase test and then further confirmed as Xoo pathogen by PCR and pathogenicity test as mentioned below. Confirmed Xoo pathogens were stored in PSA agar slants at 4°C and 20% glycerol at −80°C for further use.

Molecular confirmation of Xoo was carried out using the primers JLXooF Forward primer (5′-CCTCTATGAGTCGGGAGCTG-3′) and JLXooR Reverse primer (5′-ACACCGTGATGCAATGAAGA-3′) encoding glycosyltransferase gene. PCR cycling condition includes initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 60 s, annealing at 58°C for 45 s, extension at 72°C for 45 s, and final extension at 72°C for 7 min amplifying an internal fragment of 230 bp (Lu et al., 2014).

Rice seeds of the highly susceptible variety TN-1 were sown in a nursery bed, and 3 weeks later, seedlings were transplanted to plastic pots (size 30 cm diameter × 30 cm height). Rice plants were grown under controlled conditions. Bacterial suspensions of Xoo isolates were prepared in sterile normal saline at a concentration of 10⁸ CFU/mL and were inoculated by the leaf-clipping method (Kauffman et al., 1973) using sterile scissors in fully expanded leaves of rice plants. Control leaves were treated with sterile normal saline. Lesions on leaves were observed daily for 14 days after inoculation (Backer, 2002). Leaves showing typical symptoms of BLB were processed for re-isolation of the pathogen in PSA plates.

The Xoo pathogen isolated as mentioned earlier namely XooCG-01 was used as a host for the isolation of bacteriophages against Xoo. In addition to Xoo strains, Xanthomonas campestris isolated from cabbage infected with black rot disease and Bacillus cereus, B. subtilis, B. thuringiensis, Pseudomonas spp., and Enterobacter spp. isolated previously from Chhattisgarh at ICAR-NIBSM were used to determine the host range of phages.

Bacteriophages against Xoo were isolated by an enrichment method (van Twest and Kropinski, 2009) with slight minor modifications from rice field water, soil, and plant debris samples (n = 147) collected from the rice fields of Chhattisgarh state (119), and its adjoining seven states Jharkhand (n = 7), Uttar Pradesh (n = 6), Odisha (n = 5), Madhya Pradesh (n = 3), Andhra Pradesh (n = 2), Maharashtra (n = 2), and Telangana state (n = 2) during Kharif 2019. Approximately 5 gm of soil or 5 gm of triturated plant tissue debris samples were dissolved in 50 mL of nutrient broth and incubated at 28°C for 1 h and then centrifuged while rice field water samples were centrifuged directly to remove the soil sediments and large coarse particles. In brief, 5 mL of the log phase culture of Xoo grown in PS broth (peptone 10 g; sucrose 10 g; sodium glutamate 1 g; and distilled water 1,000 mL) was added to 10 mL of 5X strength PS Broth, and then, 35 mL of the sample supernatant was added and incubated at 28°C for 5 days. On days 2 and 5, a 25 mL aliquot of processed sample was centrifuged to clarify bacterial culture and the supernatant was filtered through a 0.22 μm PVDF filter (Merck Millipore).

For bacteriophage isolation, the filtrate was inoculated in triplicates by agar overlay technique on PSA double-layer plates containing solid (1.5% agar) and semi-solid (0.7% agar) PS medium supplemented with the bacterial host and incubated at 28°C for 5 days until the formation of plaques (van Twest and Kropinski, 2009; Jain et al., 2015). Plates having plaque formation (clear lysis, appreciable by naked eyes, on Xoo culture lawn) were preserved, and the plaques were confirmed for the presence of phage by secondary streaking. The phage plaques were picked up with a sterile toothpick and were purified by thrice successive single plaque isolation methods to achieve pure phage isolates. Pure plaques were collected with a sterile toothpick and transferred to Xoo culture broth. After incubation at 28°C for 24 h, the bacterial cells were removed through centrifugation and the supernatant was filtered through a 0.22 μm sterile filter.

Phage stocks were prepared from semi-confluent growth on several soft agar plates obtained by inoculating diluted purified phage solutions and incubated for 28°C. After 48 h, plates with a semi-confluent lawn of plaques were added with 3 mL of SM buffer and incubated at 4°C overnight (Shi et al., 2012; Jain et al., 2017). The SM buffer from all plates was collected into a 50 mL polypropylene tube and centrifuged at 7,000 rpm for 10 min to pelletize the bacterial cell. The supernatant was filtered through 0.22 μm sterile filtration units and stored at 4°C till further use.

To determine the concentration of bacteriophage in the stock filtrate, 100 μL of the filtrate was added to 900 μL of PBS buffer (pH 7.4) and 10-fold serial dilutions were made in PBS buffer. In brief, 100 μL from each dilution was subjected to plaque formation for 24 h using the double-layer agar method. The concentration of each bacteriophage in the stock filtrate was expressed as plaque forming units (PFU/mL).

The lytic activity of isolated phages was tested on six different isolates of Xanthomonas oryzae pv. oryzae, bacterial isolates of X. campestris, Bacillus cereus, B. subtilis, B. thuringiensis, Pseudomonas spp., and Enterobacter spp. isolated at ICAR-NIBSM during 2015–2020. The streak test method was used as a rapid and efficient method for determining the host range. In brief, 200 μL individual bacterial strains in the log phase were added individually to tubes containing 5 mL of 0.7% soft agar. The suspension was transferred to a Petri dish with PSA or NA and allowed to solidify. All the phage isolates were streaked individually as a single line on agar plates, which were then incubated at 28°C for 48 h. The experiment was performed in duplicate.

A drop of 3 μL purified high-titer phage (≥10⁸ PFU/mL) was deposited on a carbon coated grid and allowed to adsorb for 1 min, followed by 2% phosphotungstic acid (PTA; pH 6.8–7.0) and allowed standing for 2 min. The excess PTA was drained off, dried, and observed under a transmission electron microscope (Model: CRYO-TEM TALOS S; Make: Thermo Scientific) under 80 kV at Sophisticated Analytical Instrument Facility (SAIF), All India Institute of Medical Science (AIIMS), New Delhi, India. Images of phages were acquired using Digital FluCam Ceta 16 M Camera. The dimensions of capsid diameter and the tail length of at least six phage particles of each phage sample were measured to calculate the average and standard error values using the software ImageJ (Ver. 1.53e). Based on the TEM morphology, bacteriophages were classified into their respective families as per the guidelines of the International Committee on Taxonomy of Viruses (ICTV; Ackermann, 2011) and named following the phage nomenclature defined by Kropinski et al. (2009).

Purified phage NR08 in SM diluent having titer 6.2 × 10⁹ PFU/mL was exposed to different physicochemical conditions (pH, temperature, direct sunlight, and UV-C radiation) and then titrated against host bacteria using the soft agar overlay method. To determine the heat stability, purified phage preparation was exposed to various temperatures, viz., 4°C, 25°C, and 40°C to 90°C on a gradient thermocycler for 15 min and then titrated (Lim et al., 2013). In the pH stability assay for NR08 phage, 100 μL of phage preparation was added to 900 μL SM buffer adjusted with NaOH or HCl to a pH range of 2–12 and incubated at room temperature for 18 h and then titrated (Marquioni et al., 2022). The effect of ultraviolet (UV) irradiation was also studied, for which as a source of UV light, a UV lamp integrated into the laminar flow hood was used (Philips UV light 36 Watt G36T8, UVC = 254 nm). Phage NR08 was diluted in SM buffer to an initial titer of 10⁶ PFU/mL, and 1 mL each was poured into two Petri dishes. Phage samples were exposed to UV irradiation from a distance of 90 cm for the duration of 2 and 5 min, and then, the titer of phages in exposed plates was assayed (Zaczek-Moczydłowska et al., 2020). To determine the direct effect of sunlight on the viability of phage NR08, 1 mL the purified phage preparation (~10⁸ PFU/mL) was poured into a sterile Petri plate with cover and 20 such plates were kept in the open field under direct sunlight at 11:00 a.m. Indian standard time in the month of December 2020. At an interval of every 15 min, one plate was collected and the count of viable phages in exposed plates was assayed. Similarly, the effect of some chemicals, SDS (1%), phenol (5% aqueous), and formalin (40%) on the survivability of phages was also observed (Pandey et al., 2013). The purified phage preparation was mixed with an equal volume of the test chemical and incubated for 1 h at 28°C with intermittent shaking and then subjected to phage titration.

Percentage reduction in phage titer was calculated using the following formula:

Percentage titer reduction = (Phage original titer − Phage titer after treatment) × 100/Phage original titer

To determine the infection cycle of phage, the one-step growth curve of phage vB_XooS_NR08 was drawn following the protocol (Owen et al., 2017). One mL of phage NR08 (1 ×10⁸ PFU/mL) was added to the 10 mL of the early-log phase of the XooCG01 culture (1 ×10⁸ CFU/mL) at 0.1 multiplicity of infection (MOI) and incubated at 28°C for 5 min. The unabsorbed phages were then removed through centrifugation at 14,000 rpm for 1 min. The phage titer (unabsorbed phages) in the supernatant was determined using the double layer agar plate method to calculate the absorbance rate. Thereafter, the precipitate was reconstituted into 50 mL of fresh PSB and cultured at 28°C. One mL of sample was withdrawn at the interval of every 10 min and centrifuged at 14,000 rpm for 1 min. The supernatants were processed for phage count at each time point to evaluate the count of free phage using the double-layer agar plate method.

The adsorption rate of phage was calculated as follows:

Adsorption rate = (Initial phage titer − unabsorbed phages titer)/Initial phage titer

A one-step growth curve was plotted, and the latent period, rise period, and phage burst size of phage NR08 were calculated as previously described by Owen et al. (2017). The latency period is the time from the adsorption of the phage on bacterial cells to the release of new phages from the host bacterial cells. The burst period refers to the period from the beginning of phage release after latency to the end of phage release. The burst size was calculated as the ratio of the final count of released phage particles to the initial count of infected bacterial cells at the time of infection.

The structural proteins of vB_XooS_NR08 phage were separated and observed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). High-titer phages were precipitated overnight at 4°C using 30% polyethylene glycol (PEG-8000) and 3 M NaCl in the ratio of 2:1 and then centrifuged at 14,000 rpm for 15 min. The pellet was reconstituted in SM buffer to concentrate one-tenth of the initial volume of phage and then subjected to SDS-PAGE as per the method of Laemmli (1970) using 10% resolving gel of SDS-PAGE and Tris-glycine as running buffer. The concentrated phage samples were mixed with 2X sample buffer and boiled at 100°C in a water bath for 10 min. A standard broad range protein molecular weight marker (Promega) with a range of 10 to 210 kDa was used to estimate the molecular weights. The gel was stained with Coomassie Brilliant Blue R250 dye and destained with the destaining solution for 30 min, and then, the gel was visualized and documented using the Gel Doc system (Protein simple, Alfa Innotech Corporation, United States).

To remove bacterial DNA and RNA contamination prior to phage genome recovery, high-titer NR08 phage suspension (~10⁸ PFU/mL) was treated with DNase I and RNase A for 2 h at 37°C. Thereafter, both the enzymes were inactivated by treating with EDTA at room temperature for 10 min followed by proteinase K treatment for 2 h at 55°C. High-titer phage was precipitated using 30% polyethylene glycol (PEG-8000) and 3 M NaCl as discussed for protein profiling. The PEG-concentrated phage pellet suspended in SM buffer was used for phage nucleic acid extraction using Wizard Genomic DNA Purification Kit (Promega) in accordance with manufacturer protocols. Nucleic acid integrity and absence of degradation were verified by 0.8% agarose gel electrophoresis. Furthermore, nucleic acid was treated with DNase I, RNase A, and S1 nuclease to identify the type of nucleic acid present in the NR08 phage.

The quality and quantity of isolated phage DNA were analyzed on a high-sensitivity DNA electrophoresis chip run on Agilent Bioanalyzer 2100 (Agilent Technologies, United States). The genome sequencing library was prepared for pair end sequencing using Illumina HiSeq following the manufacturer protocol, and qualitative and quantitative analyses of the library were performed using an Agilent Bioanalyzer 2100 (Agilent Technologies, United States). The phage’s genomic DNA libraries were sequenced using the Illumina sequencing approach on Illumina HiSeq 2500 machine at Nucleome Informatics Pvt. Ltd., Hyderabad. The raw reads generated by sequencing of phage genomic libraries were cleaned and quality-trimmed using the FASTP software (Chen et al., 2018). The high-quality filtered reads of phage were used for the de novo assembly using the SPAdes assembler v3.14 (Bankevich et al., 2012) using the “careful mode” which includes reading correction module, reduces the mismatches and false indels arising during the assembly and k-mer-titration based on read characteristics. The de novo assembly was also performed using SKESA assembler v2.3.0 (Souvorov et al., 2018) with default parameters. The assemblies are finally chosen on the basis of the following parameters: (i) N50; (ii) number of contigs; (iii) maximum contigs size; and (iv) number of bases in the assembly. The assembly generated by SPAdes produced more N content and gaps, while the assembly produced by SKESA-assembler did not contain N and gap. Therefore, SKESA-assembled genome assembly was further used for annotation and analysis.

The assembled contigs were used for the functional annotation using RASTtk (Overbeek et al., 2014; Brettin et al., 2015), and tRNA detection was performed using the software tRNAscan-SE v. 2.0 (Schattner et al., 2005) and ARAGORN (Laslett and Canback, 2004) using with default settings. Functional annotation was also performed using Blast2GO 6.0.3 (Conesa et al., 2005). The predicted genes were used to search the NCBI non-redundant database, the conserved domain database, the Cluster of Orthologous Groups database, and the InterPro database and were annotated using Blast2GO V.0.3. Automated annotation performed by RASTtk was manually curated by individually analyzing each predicted gene using Blast2GO. Phage life style predication was performed using the online tool phage AI¹ to predict phage lifestyles.

The assembled genome of Phage NR08 was compared with the NCBI databases using BLASTn (Altschul et al., 1990). The blast was done using BLASTN 2.12.0⁺ (Zheng et al., 2000) with NCBI databases, and the results were visualized using the Kablammo: an interactive, web-based BLAST results using http://kablammo.wasmuthlab.org. Comparative genome analysis was conducted by whole-genome alignment with 12 previously reported Xoophages having genome sequences available with NCBI. Genome sequences of all the previously reported Xoophages were downloaded which includes 10 Xoophages namely XPP/XPV (Accession nos. MG944227 to MG 944236; Kovács et al., 2019), OP2 DNA (Acc. no. AP008986; Inoue et al., 2006), and Xoo-sp2 (Acc. No. KX241618; Dong et al., 2018) and aligned using CLC Genomics Workbench V 20.0.4 with the default parameters and visualization of comparative map and genome map of NR08 phage. Average nucleotide identity comparison 1.0 was performed with a default setting minimum similarity fraction of 0.8 and minimum length fraction of 0.8.

To define the evolutionary history of the new phage species, a phylogenetic analysis was performed. Nucleotide sequences of NR08 phage were compared with the NCBI database using BLASTn. The whole-genome data of phages having the highest homology with NR08 were downloaded from NCBI, and a phylogenetic tree was constructed using the neighbor-joining method using Jukes-Cantor nucleotide distance measure with 500 bootstrap replicates using CLC Genomics Workbench v. 20.0.4.

One mL of XooCG01 (10⁸ CFU/mL) at exponential growth phase was co-inoculated with 1 mL of NR08 phage (10⁷ PFU/mL) at 0.1 MOI in a culture flask into 50 mL of PSB, while another flask, with the same volume of PSB, inoculated with 1 mL of XooCG01 (10⁸ CFU/mL) only, was kept as bacterial control. Both the flasks were incubated at 28°C for 48 h. At every 6-h interval, 500 μL of the medium was taken out from both the flask and processed for total colony count of host bacteria by 10-fold serial dilution method and plated in PSA plates in triplicate and incubated at 28°C for 72 h. The total bacterial colony count of every collect was evaluated in terms of CFU/mL for both bacterial control and phage-treated. The final bacterial concentrations were compared between phage inoculated and bacterial control cultures to determine the effect of phage in reducing bacterial growth. The percent decrease in total bacterial count in phage-treated was calculated as compared to the bacterial control.

To determine the protective efficacy of vB_XooS_NR08 phage against bacterial leaf blight infection, rice seedlings were transplanted in pots (size 30 cm diameter × 30 cm height) having five plants in each pot and each treatment was replicated three times. The experiment was arranged in a completely randomized block design at environmental temperature. At the maximum tillering stage, rice plants were infected with Xanthomonas oryzae pv. oryzae pathogen at a concentration of around 10⁸ CFU/mL using the leaf clip inoculation method in highly susceptible TN-1 variety as previously described (Kauffman et al., 1973). Briefly, the 3-day-old grown XooCG01 culture on PSA was harvested in sterile saline solution, and suspension turbidity was adjusted to obtain a concentration of 10⁸ CFU/mL to infect the rice plants by clipping five leaves per plant. After 72 h of BLB infection, plants were treated with a single application of selected phage in two preparations: (i) neat NR08 phages (without any supplement or additive) at concentration 10⁷ PFU/mL and (ii) NR08 phage at concentration 10⁷ PFU/mL supplemented with 2% skim milk preparation, using the spray method, whereas the phage untreated plants were sprayed with sterile water and considered as untreated infected control plants. Application of phage preparation was performed with a handheld sprayer during the evening hours of the day. The plants were observed and recorded daily for up to 21 days for the progression of disease symptoms in both the phage-treated and the untreated control groups. The length of lesions on 10 leaves per treatment was recorded and evaluated for each group and used for analysis. The disease suppression efficacy of the phage-treated group was compared with the untreated control group.

The data were subjected to analysis of variance (ANOVA) using the online Indian NARS statistical computing portal at the IASRI server.² Statistical significance was evaluated using Duncan’s multiple range test (DMRT), and the p-value <0.01 was considered significantly different.

The leaves showing typical symptoms of bacterial blight were collected from rice fields in five states of India. Twenty-four bacterial isolates were obtained from the infected leaf samples on PSA plates with typical characteristics of Xoo, i.e., yellow, round, mucoid colonies convex appearing with a smooth margin. Isolates with Gram-negative, KOH positive, oxidase negative, and catalase positive reactions were further confirmed as Xoo pathogens by PCR and pathogenicity test. All 24 isolates were further subjected to Xoo-specific PCR and pathogenicity test. Six isolates were confirmed as Xanthomonas oryzae pv. oryzae which produced 230 bp PCR product using Xoo-specific PCR primers and showed typical symptoms of BLB in pathogenicity test in rice. In the pathogenicity test, on 5 days post-inoculation, symptoms started appearing with the formation of water soaked lesions from the margin, and, by 10 dpi, lesions were produced all along the leaf blade in a wavy pattern extending in length and width (Figure 1). These six isolates belonging to states Chhattisgarh (3), Odisha (2), and Tripura (1) were named XooCG01, XooCG02, XooCG03, XooOD01, XooOD02, and XooTR01. Among these six isolates, XooCG01 showed maximum pathogenicity in terms of the production of leaf lesions, disease severity, and wilting of plants. Therefore, the XooCG01 strain was used as the host strain for phage isolation and further experiments.

Water, soil, and plant debris present in rice fields provide an enriched composition of the bacterial population, making it an excellent environment for the isolation of bacteriophages. Out of 147 samples collected, we isolated and purified 19 bacteriophages of Xanthomonas oryzae pv. oryzae from rice field water (11), soil (6), and field debris (2) using phage enrichment protocol representing three states of India, viz., Chhattisgarh (17), Madhya Pradesh (1), and Telangana state (1). These isolated Xoophages were designated as NR01 to NR19 (Table 1). The maximum recovery of phages was from samples belonging to Xoo-infected field, while only one phage NR14 was isolated from uninfected field water. These phages produced clear round lytic plaques of size ranging from 1 to 10 mm in diameter (Figure 2). Based on the plaque size, phages were grouped into three categories, viz., small size plaque producers (1–3 mm), medium size plaque producers (3–6 mm), and large size plaque producers (6–10 mm) containing eight, seven, and four phages, respectively. Among all phages, the smallest size plaques were observed in phages NR08 and NR09, while the largest plaques were formed by phage NR11. The titer of the isolated phages varied from 3.4 × 10⁶ to 6.2 × 10⁹ PFU/mL with the lowest and highest in NR14 and NR08 phages, respectively (Table 1).

All phages were able to lyse Xoo strains and displayed the same host range, showing the lytic activity for tested five Xoo isolates namely XooCG01, XooCG02, XooCG03, XooOD01, and XooOD02 except XooTR01, forming clear zone around the phage streaked lines on the bacterial lawn of each tested strain, respectively, while none of the phages showed any lytic activity for X. campestris and other heterologous host bacterial species used in this study, viz., Bacillus cereus, B. subtilis, B. thuringiensis, Pseudomonas spp., and Enterobacter spp. (Table 1) suggesting the isolated phages were highly specific to their bacterial host Xoo.

The host specificity of phages revealed that all the 19 isolated Xoophages infected all five Xoo strains isolated from the state of Chhattisgarh and Odisha, while only four phages (phages NR08, NR12, NR15, and NR18) possess infectivity for Xoo strain belonging to state Tripura. This might be due to variation in the binding capability of phages to host XooTR01 strain isolated from the northeastern state of India, whereas none of the phages were able to infect the bacteria other than Xoo indicating their high host specificity and, thus, will not harm the beneficial microflora present in the soil when further used as a biocontrol agent against Xoo. Phage NR08 has a broad range of Xanthomonas host strains, as it infected all the six strains of Xoo tested and was also found to have the highest titer, hence, the phage NR08 was used for further studies.

On transmission electron microscopy, all 19 Xoophage isolates revealed head and tail morphology belonging to the order Caudovirales with representatives of different morphotypes families, viz., Myoviridae (4), Siphoviridae (12) Podoviridae (2), and unclassified (1) indicating that a substantial diversity of phages was isolated during this study. Among the four myoviruses, having a contractile tail, the head width ranged from ~52 to ~64 nm, head length ranged from ~55 to ~68 nm, and tail lengths varied between ~96 and ~190 nm with A1 morphotype. In few electron micrographs of phages, the contractile stage of the phage tail was also observed. Analysis of 12 siphoviruses, having a non-contractile tail, revealed head width ranged from ~51 to ~98 nm, head length ranged from ~58 to ~122 nm, and tail length ranged from 132 to ~212 nm. Out of 12 siphoviruses, morphotypes B1 and B2 were observed in six phages each. While, in the two podoviruses, having a non-contractile short stubby tail, the head width ranged from ~32 to ~44 nm, head length ranged from ~36 to ~45 nm, and tail length ranged from ~12 to ~15 nm with C1 morphotype (Figure 3 and Table 2). In some of the siphoviruses and the myoviruses phages, tail tubes, tail plates, and tail filaments were also visible. Based on the TEM morphology, bacteriophages were classified into their respective families as per the guidelines of the International Committee on Taxonomy of Viruses (ICTV; Ackermann, 2011) and named following the phage nomenclature defined by Kropinski et al. (2009).

Phage stability was assessed for various environmental and chemical factors. NR08 phage was 100% viable at the temperature range of 4°C–40°C, up to 30% viable at 60°C, while less than 1% viability was seen above 70°C indicating the loss of phage viability at elevated temperatures (Figure 4A). Similarly, when tested for pH stability of phage NR08, more than 80% of phages were viable at pH 5–9 as indicated by the remaining phage titer after exposure to this pH range. The phage titer was drastically reduced when incubated at pH below 5 or above 9 as indicated by no detectable effect on bacteriophage survival and infectivity (Figure 4B).

Phage NR08 when tested for UV radiation sensitivity, on exposure duration of 2 and 5 min, the reduction of active phages was up to 6.2 ×105 PFU/mL and almost 100%, respectively. Phage NR08, when exposed to direct sunlight, more than 75% of phages can survive up to an exposure time of 135 min, and thereafter, the viability drastically reduces to less than 1% in exposure duration of 180 min (Figure 4C). Furthermore, all phages were highly sensitive to 40% formalin and 1% SDS showing less than 1% viability, while less than 10% phages were viable after exposure to 5% aqueous phenol.

To enumerate the adsorption rate, phage latent period, burst period, and burst size, we determined a one-step growth curve. Approximately 93% of the NR08 phage particles were adsorbed on the XooCG01 cells in 5 min at 0.1 MOI. The one-step growth curve showed that the phage NR08 had a latent period of 40 min followed by a burst period between 40 and 70 min (Figure 4D). At the end of the burst period, the burst size was approximately 250 virions per infected cell.

Purified phage vB_XooS_NR08 particles were denatured and separated using SDS-PAGE. At least nine distinct protein bands of approximate size 10.9, 17.3, 32.8, 40, 46.6, 55, 77.4, 106, and 166.4 kDa were observed in the Coomassie-stained SDS-PAGE gel, which were expected to be structural proteins of vB_XooS_NR08 by their estimated molecular weights. The predicted proteins may be phage tail fiber proteins, phage tail length tape measure protein 1, phage tail length tape measure protein H, phage DNA polymerase, phage structural protein, phage portal protein, minor capsid protein, phage capsid, and scaffold protein (Figure 5).

The nucleic acid of phage NR08 was extracted, and isolated nucleic acid was treated with DNase I, RNase A, and S1 nuclease to identify the nature of nucleic acid which revealed it to be double-stranded DNA. The Illumina sequencing of bacteriophage vB_XooS_NR08 genome generated a total of 16,539,460 pair end reads with a total base of 2662.85 Mb and with an average read length of approximately 161 bp. After quality control and filtering, the total reads remain 12,264,972 with a total base of 1689.24 Mb and average read length of 137.7 bp having Q30 bases of 95.58 and GC content of approximately 52.51%. The detail of sequencing read statistics of raw reads and filtered reads of the phage genome is mentioned in Table 3. The filtered reads were taken for de novo assembly using the SKESA and SPAdes genome assemblers. The quality check of contigs showed that the assemblies generated by SPAdes had large gaps and N string, thus, it was not taken for further analysis. The SKESA genome assembler produced a total of 39 contigs with a size of 98,812 bp and a G + C content of 52.9%. The detail of the de novo assembly of whole-genome sequences of NR08 using the SKESA assembler is given in Table 3. The complete genome sequence of bacteriophage vB_XooS_NR08 was deposited in GenBank under accession number OQ055246, and the raw sequence reads were deposited in the NCBI database under the BioProject ID: PRJNA844261, BioSample number: SAMN29042521, and SRA accession number: SRR19757545.

The whole-genome sequencing analysis of phage NR08 showed the double-stranded linear DNA of 98,812 bp in size with a G + C content of approximately 52.9%. The NR08 genome contains 142 open reading frames (ORFs; Figure 6) and is larger in size than those of previously reported Xanthomonas phages (Ogunyemi et al., 2019; Liu et al., 2021; Nazir et al., 2021). The genome assembly was screened for the presence of tRNA using the software tRNAscan-SE v. 2.0 which showed the presence of only one tRNA namely trna1-GlnTTG of 72 bp. Out of 142 ORFs, 83 were present on the positive sense strand while 59 were found on the negative sense strand of the phage genome. Phage lifestyle prediction was done using the phageAI online tool which showed the nature of phage to be a virulent having lytic lifestyle.

The functional annotation of the genome sequence revealed that the NR08 phage has 141 probable protein-encoding genes and one gene for tRNA^Gln as depicted in Figure 6. Out of 141 protein coding ORFs, 23 ORFs were predicted to code genes for phage structural and host interaction proteins which include virion proteins, major capsid proteins, major tail tube proteins, minor tail proteins, head–tail joining proteins, tail fiber proteins, and tape measure proteins, whereas 42 ORFs have been annotated as hypothetical proteins. Furthermore, bioinformatics analysis revealed that 76 ORFs code for functional proteins of which 32 ORFs were associated with phage metabolism and were involved in DNA replication, recombination, repair, transcription, translation, and other related nucleotide and protein metabolism. Similarly, eight ORFs were related to host lysis function, 12 ORFs were associated with phage assembly and packaging function, and eight ORFs encoding phage portal proteins, chaperones, and scaffold proteins were involved in phage morphogenesis. While 12 ORFs were having endonuclease activity of which seven were having HNH endonuclease function, whereas four ORFs codes for the domain of the unknown function (DUFs domain containing proteins). Out of eight genes associated with host lysis, three ORFs are for holin proteins, two for phage spanin Rz, and one each ORF codes for lysozyme, Clp protease, and peptidoglycan endopeptidase. The complete genome was categorized into four modules based on the functions of encoded proteins in the virion, viz., DNA packaging module, morphogenesis module, DNA replication module, and lysis modules (Figure 6; Supplementary Table 1). Among the 141 ORFs with a predicted structure or function, none were associated with phage lysogenic cycle, antibiotic resistance, toxicity, and allergens.

The whole-genome sequence of vB_XooS_NR08 was analyzed using BLASTN analysis against the NCBI non-redundant DNA database. A nucleotide comparison with available genome sequences in the NCBI database showed that out of the top 13 hits, the NR08 phage showed the highest genome similarity with Pseudomonas phage PaMx42 Acc. No. JQ067092 (40% query coverage, 95.39% identity, and acc. Length 43,225 bp), Xanthomonas phage Samson, Acc. No. MN062187 (40% query coverage, 96.68% identity, and acc. Length 43,314 bp), and Pseudomonas phage Guyu, Acc. No. MZ927746 (40% query coverage, 93.74% identity, and acc. Length 43,141 bp). To explore further the evolutionary position of the NR08 phage, a phylogenetic analysis was performed with MEGA using the neighbor-joining method for phage NR08 and its related 12 different phages. The phylogenetic tree showed two major branches based on the whole-genome analysis of these phages indicating NR08 is closely related to three phages, viz., Pseudomonas phage PaMx42, Xanthomonas phage Samson, and Pseudomonas phage Guyu (Figures 7A,B).

The comparative genomic analysis of NR08 phage with previously reported 12 Xoophages was conducted which includes 10 Xoophages namely XPP/XPV (Accession no. MG944227 to MG 944236; Kovács et al., 2019), OP2 DNA (Acc. No. AP008986; Inoue et al., 2006), and Xoo-sp2 (Acc. No. KX241618; Dong et al., 2018). The genome size of these reported Xoophages ranged from 45 to 47 kb except phage Xoo-sp2 which has a genome size of 60.3 kb in length, while the NR08 phage isolated in this study had a genome size of 98.8 kb.

Average nucleotide identity (ANI) was calculated and found that the ANI was 83.95 with (MG994227-29, 31-33) and 83.44 with AP008986, least or no ANI with (MG994230, 34-36, and KX241618). The average alignment percentage (AP) of NR08 with other Xoophages ranged from 1.19 to 1.25 for MG944227-29, MG944231-33, and AP008986 and 0.70 to 0.71 for MG944230 and MG944234-36 and least AP 0.32 for KX241618 phage (Figures 8A,B). This shows that the most of genome sequence information is novel for bacteriophage, vB_XooS_NR08, infecting Xanthomonas oryzae pv. oryzae. The genome comparison showed that the matched genome sizes were approximately 43–44 kb out of the 98.8 kb genome size of NR08.

KEGG mapping of the phage genome revealed that it codes for the enzymes of different pathways namely purine metabolism; ubiquinone and another terpenoid-quinone biosynthesis; oxidative phosphorylation; pyrimidine metabolism; and thiamine metabolism. The name of enzymes and their respective pathways are as follows: (i) EC: 3.6.1.15—phosphatase enzyme involved in purine metabolism and thiamine metabolism pathways; (ii) EC: 1.6.5.2—dehydrogenase (quinone) enzyme of ubiquinone and other terpenoid-quinone biosynthesis pathways; (iii) EC:3.5.4.12—deaminase enzyme of pyrimidine metabolism pathway; and (iv) EC:7.1.1.2—reductase (H + -translocating) of oxidative phosphorylation pathway (Supplementary Figure 1).

The bacterial challenge assay of NR08 on its host bacteria was tested by evaluating the change in total colony count of host bacteria after NR08 infection at every 6-h interval up to 48 h. NR08 phage was effective against the host strain in the tested 0.1 MOI, and almost no bacterial growth was observed in the initial 24 h after co-inoculation. After 24 h, the minimal steady growth of XooCG01 occurred indicating that the equilibrium between cell growth and lysis has been achieved (Figure 9A). The phage NR08-treated bacterial count showed bacteriostasis up to 24 h; thereafter, up to the studied period of 48 h, there was more than 3 log₁₀ reduction in the bacterial count, i.e., 99.95% reduction as compared to untreated bacterial control (Figure 9B). These results suggest that NR08 can be explored as a potent phage candidate for biocontrol of bacterial blight infection.

In the in-planta pot experiments for the effectiveness of selected phage against BLB pathogen, the treatment includes a single application of phage preparations 72 h after the bacterial inoculation, while infected untreated rice plants were used as control. Application of treatments was performed with a handheld sprayer during the evening hours of the day. Plants were assessed for disease severity on 7, 14, and 21 days post-infection or 4, 10, and 18 days post-treatment. Data sets of measured lesion lengths from 10 observations per group were used for analysis. Phage-treated rice plants showed a significant reduction of BLB symptoms as compared to untreated control plants (Figures 10A,B). Within the first 3 weeks of observation, the disease leaf lesion length was recorded as lowest in the NR08 phage treatment with a mean length of 1.02 ± 0.20 cm, 1.74 ± 0.32 cm, and 3.43 ± 0.63 cm at 7, 14, and 21 dpi, respectively, followed by the NR08 phage preparation supplemented with 2% skim milk showing leaf lesion length of 2.15 ± 0.62 cm, 5.53 ± 0.96 cm, and 9.08 ± 0.99 cm at 7, 14, and 21 dpi, respectively, and were significantly different at p < 0.01 from untreated infected control showing leaf lesions length of 10.45 ± 1.77 cm, 15.1 ± 2.91 cm, and 16.55 ± 3.02 cm at 7, 14, and 21 dpi, respectively (Table 4; Figure 10). The NR08 treatment provided the highest disease control efficacy ranging from 79.27% to 90.23% at different dpi followed by NR08 supplemented with SM treatment at 45.13% to 79.42% at different dpi, between which there was a significant difference. In both the experimental phage treatment groups, the application of phage NR08 treatment significantly reduced the lesion on rice leaves as compared to the untreated infected control (Table 4).