Gamma-induced mutants of Bacillus and Streptomyces display enhanced antagonistic activities and suppression of the root rot and wilt diseases in pulses.

This study aims to increase Bacillus and Streptomyces antagonistic activity against the root rot and wilt diseases of pulses caused by Macrophomina phaseolina and Fusarium oxysporum f. sp. udum, respectively. To increase antagonistic action, Bacillus subtilis BRBac4, Bacillus siamensis BRBac21, and Streptomyces cavourensis BRAcB10 were subjected to random mutagenesis using varying doses of gamma irradiation (0.5-3.0 kGy). Following the irradiation, 250 bacterial colonies were chosen at random for each antagonistic strain and their effects against pathogens were evaluated in a plate assay. The ERIC, BOX, and random amplified polymorphic studies demonstrated a clear distinction between mutant and wild-type strains. When mutants were compared to wild-type strains, they showed improved plant growth-promoting characteristics and hydrolytic enzyme activity. The disease suppression potential of the selected mutants, B. subtilis BRBac4-M6, B. siamensisi BRBac21-M10, and S. cavourensis BRAcB10-M2, was tested in green gram, black gram, and red gram. The combined inoculation of B. siamensis BRBac21-M10 and S. cavourensis BRAcB10-M2 reduced the incidence of root rot and wilt disease. The same treatment also increased the activity of the defensive enzymes peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase. These findings suggested that gamma-induced mutation can be exploited effectively to improve the biocontrol characteristics of Bacillus and Streptomyces. Following the field testing, a combined bio-formulation of these two bacteria may be utilised to address wilt and root-rot pathogens in pulses.


Introduction
Pulses are India's most important protein dietary source, with minimal fat and high fibre and nutrient content [1]. India cultivates roughly 34% of the worldwide cultivable land for pulses and contributes 24% of the global pulses production [2]. However, productivity of pulses in India is extremely low (652 kg ha −1 ) (FAOStat, 2013). It is estimated that wilt and rot diseases cause 40-45% of the yield loss in pulses [3,4]. Fusarium oxysporum f. sp. udum and Macrophomina phaseolina are the primary pathogens responsible for wilt and root rot diseases in pulses, respectively. Traditional methods for controlling soil-borne fungal infections include fungicide treatment, fumigation, and crop rotation [5,6]. However, the use of fungicides has greater drawbacks, such as environmental pollution, negative impact on soil-beneficial bacteria and insects, and development of disease resistance to the specific fungicide [7,8]. As a result, alternate sustainable and environmentally beneficial solutions are required.
Natural microbial strains produce only trace levels of antifungal compounds. This is because they require these secondary metabolites for competitive advantage and do not overproduce them. Microorganisms have evolved regulatory systems that allow a strain to prevent overproduction of its metabolites. Hence, strain enhancement programmes are essential for commercial application [30]. The use of random mutagenesis to modify strains aids in the improvement of biocontrol abilities and/or the synthesis of antifungal metabolites. Chemical or physical mutagens (ultraviolet or gamma irradiation) are employed [31]. Gamma irradiation boosted Bacillus subtilis secondary metabolites and disease control abilities [32,33], Trichoderma harzianum protease activity [34][35][36], Streptomyces antibiotic synthesis [37][38][39], and Bacillus thuringiensis chitinase production [40].
B. subtilis BRBac4, Bacillus siamensis BRBac21, and Streptomyces cavourensis BRAcB10 isolated from the pulses rhizosphere previously demonstrated numerous plant growthpromoting features as well as antagonistic activity against both the fungal diseases, F. oxysporum f. sp. udum and M. phaseolina (unpublished data). Random mutagenesis was performed in B. subtilis BRBac4, B. siamensis BRBac21, and S. cavourensis BRAcB10 using gamma irradiation and the mutation was validated by ERIC, BOX, and random amplified polymorphic DNA (RAPD) studies. We then tested these effective mutants against F. oxysporum f. sp. udum and M. phaseolina in vitro and in pot culture in pulses.

Materials and methods
Microorganisms and growth conditions B. subtilis BRBac4 (GenBank: MN165562), B. siamensis BRBac21 (GenBank: MN165655), and S. cavourensis BRAcB10 (GenBank: OK427229) were isolated from pulses rhizosphere soils and used for this study. Bacillus spp. and Streptomyces were grown in an incubator shaker at 28 ± 2°C in liquid nutrient and tryptic soya media, respectively. F. oxysporum sp. udum NAIMCC-F-01125 was obtained from the Indian Council of Agricultural Research, Maunath Bhanjan, Uttar Pradesh, India, and M. phaseolina BRMP1 (GenBank: MN165654) was previously isolated from disease-infected pulses and obtained from the Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, India. At 28 ± 2°C, the fungal pathogens were cultured in potato dextrose agar medium.

Gamma-induced mutation
The mutation was carried out at the Gamma Chamber 5000 at the Board of Radiation and Institute of Technology, Department of Atomic Energy (DAE), in Mumbai, India. The mutation was carried out at the Gamma Chamber 5000 at the Board of Radiation and Institute of Technology, Department of Atomic Energy (DAE), in Mumbai, India as described by Afsharmanesha et al. [32]. Bacterial strains previously cultivated in liquid media were placed in cryovials in triplicate and directly exposed to various gamma irradiation dosages, namely 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 kGy. C60 had an efficiency of 2.2 kGy. Following irradiation, the bacteria were serially diluted up to 10 −6 in 0.8% saline and plated on their respective growth medium. Mutant colonies were chosen based on the physical differences between them and their wild-type counterparts. All the Bacillus and Streptomyces colonies that were chosen as putative mutants were kept in their respective growth medium for further testing. and the bacterial stain was streaked immediately opposite the mycelial disc. As a control, a fungal disc with no bacterial inoculation was used. All of the plates were incubated for 7 days at 28 ± 2°C. The following formula was used to compute the percentage of fungal growth: where Cmycelial growth in control, Tmycelial growth in the treatment plates.
DNA isolation, rep-polymerase chain reaction (PCR), and RAPD The Hi-GenoMB Miniprep kit (Hi Media, India) was used to isolate DNA from wild and putative mutant Bacillus and Streptomyces strains. Extracted DNA was electrophoresed on 0.8% agarose gel and visualised in the Gel documentation unit (Vilber, Germany). For mutation confirmation, RAPD, ERIC, and BOX amplifications were performed [34]. The PCR mixture contains two PCR master kits (Sigma, India), 10 pmol of each primer (Table S1) (BioKart, Bengaluru), and 10 ng of genomic DNA. Initial denaturation at 95°C for 5 min was followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 45°C (ERIC), 46°C (BOX), and 33°C (RAPD) for 1 min, and final extension at 72°C for 1 min. In 1% agarose gel, amplicons were electrophoresed.

PCR amplification of the antibiotic resistance gene
Two Bacillus and mutant colonies with significant antifungal activity were examined for the presence of lipopeptide genes (surfactin and iturin) [42]. Lipopeptide gene PCR amplification began with a 5 min denaturation at 95°C, followed by 30 cycles of denaturation (1 min at 94°C), annealing (1 min at 55°C), extension (1 min at 72°C), and final extension for 10 min at 72°C. Table S1 lists the primers utilised for the various lipopeptide genes.

Plant growth-promoting (PGP) traits
Indole acetic acid (IAA) The synthesis of IAA was quantified using the previously reported method [43]. Overnight developed cultures were inoculated in 5 mL of nutrient broth containing 0.1% tryptophan and cultured for 72 h at 28 ± 2°C. The supernatant was then collected for 10 min at 12,000 rpm. Around 500 µL of supernatant was mixed with 50 µL of 0.1 mM ortho phosphoric acid and 2 mL of Salkowski reagents and incubated in the dark for 30 min to generate red colour. IAA was measured using a spectrophotometer at 530 nm (Systronics ® , Spectrophotometer 166) and represented as µg mL −1 .

Bacterial motility assays
The swimming and swarming motility of the bacterial strains B. subtilis BRBac4, B. siamensis BRBac21, and their mutations was measured using the method published previously [44]. For the swimming and swarming motility assays, Luria Bertani (LB) plates were altered with 0.3 and 0.7% agar, respectively. The plates were incubated for 18 h at 28 ± 2°C and the colony diameter was measured.

Phosphate solubilisation
In a plate assay, the selected Bacillus, Streptomyces, and their mutants were examined for phosphate solubilisation efficiency. Overnight grown bacterial strains were inoculated (1 × 10 7 CFU mL −1 ) into Pikovskaya's agar medium and incubated for 48 h at 28 ± 2°C. Phosphate solubilisation was thought to be positive in the clear zone surrounding the bacterial colonies.

Hydrolytic activity
Chitinase activity Chitinase activity was measured using the previously reported method [45]. The bacterial strains were cultivated in liquid nutrient medium enriched with 0.3% colloidal chitin and shaken for 24 h at 28 ± 2°C (100 rpm). The crude enzyme was then collected by centrifugation at 10,000 rpm for 20 min at 40°C. The reaction solutions were incubated at 35°C for 25 min with 0.3% colloidal chitin (0.1 mL), a crude enzyme solution (0.1 mL), and 0.1 M McIIvaine buffer of pH 6.0 (0.2 mL). The reaction was stopped by adding 2.0 mL of 1.5 mmol potassium ferricyanide reagent. A spectrophotometer (Systronics®, Spectophotometer166) was used to measure the final absorbance at 420 nm. The enzyme activity was measured in micromoles of N-acetyl glucosamine per minute.

Protease and cellulase activity
The skim milk agar medium was used to test the protease activity of Bacillus, Streptomyces, and their mutants.
Overnight developed bacterial cultures were spotted (1 × 10 7 CFU mL −1 ) on skim milk agar medium and incubated for 72 h at 28 ± 2°C. The presence of a clean zone around the colonies suggested the presence of protease activity. To assess cellulase activity, nutrient agar medium was supplemented with 1% carboxy methyl cellulose instead of glucose, and 10 µL (1 × 10 7 CFU mL −1 ) of bacterial strains were spot inoculated. The plates were incubated for 72 h at 28 ± 2°C. Following the incubation period, the hydrolysis zone was seen by flooding the plates with 0.1% iodine solution for 15-20 min, after which the plates were rinsed with distilled water and destained with 1 M NaCl. The presence of translucent hydrolytic zones around the colonies showed the presence of positive cellulase activity [46].

Analysis of the volatile compounds
The volatile organic compounds (VOCs) of mutant Bacillus and Streptomyces strains were investigated using gas chromatographymass spectrometry (GC-MS) and a thermal desorber (TD). In a 500 mL Erlenmeyer flask, the bacteria were inoculated into liquid nutrient medium. With the help of a rubber cork, a tenax-coated stainless-steel column was fitted with the flask. The VOCs collected by the column were analysed using TD-GC-MS on a TurboMatrix 150 (Perkin Elmer, USA). For separation, a non-polar capillary column (30 m, 250 µm) coated with 5% phenyl methyl siloxane was utilised. The following temperature regime resulted in volatile separation: the initial oven temperature of 50°C was increased to 250°C at a rate of 10°C per min; the injection port was kept in split mode with a 1:10 split ratio; and the carrier gas was helium at a constant flow rate of 0.7 mL min −1 . The MS settings were as follows: full scan mode; scan range of 0.2 s at intervals of 0.1 s; electron impact spectra of 70 eV; and positive ion mode. By comparing the collected mass spectra to the NIST/EPA/NIH Mass Spectral Library, NIST 14, the VOC was identified. The approach described by Passari et al. [47] was used for VOC detection and analysis.

Effect of antagonistic mutants on suppression of root rot and wilt pathogens
Under pot culture conditions, the best-performing Bacillus and Streptomyces mutants were tested against the wilt and root rot pathogens. Red gram cv., Co (Rg)-7, Green gram cv., Co (Gg)-7, and Black gram cv.,Vamban 8 seeds were received from the Department of Pulses at Tamil Nadu Agricultural University in Coimbatore, India. The seeds were surface cleaned with 0.1% HgCl 2 and washed four times with sterile distilled water before soaking for 30 min in Bacillus (10 8 CFU mL −1 ) and Streptomyces (10 8 CFU mL −1 ) mutants produced in their respective media. For pot culture study, soil and sand were taken in the ratio of 3:1 and 2 kg of this mixture were filled in a pot (18 cm outer diameter and 20 cm height) and 5 seeds were sown in each pot. In total, five pots were maintained for each treatment and each treatment was replicated four times. Treatment details in this . Oatmeal medium was used to produce M. phaseolina inoculum. Mycelial discs were injected into the sterile oat meal medium and grown at 30°C. Before sowing the seeds of black gram and green gram, the inoculum (0.1% w/w) was combined. Inoculum of F. oxysporum f. sp. udum was made by adding 150 mL (2 × 10 8 spores mL −1 ) of broth per kilogram of soil and incubating for 7 days for spore germination. This inoculum was combined before to the sowing of red gram [48]. The plant defence enzyme was analysed 28 days after the seeds were sown (DAS). Plant samples were uprooted and taken to the laboratory for biometric measures after 35 DAS.

Enzyme analysis
After collecting 28 DAS leaf samples, they were pulverised with liquid nitrogen in a pre-chilled pestle and mortar. One gram of powdered leaf sample was combined with 2 mL of 0.1 M sodium phosphate buffer (pH 7.0), and the supernatant was collected after 15 min of centrifugation at 14,000 rpm at 4 C. The enzyme activity of peroxidase (PO), polyphenol oxidase (PPO), and phenylalanine ammonialyase (PAL) were determined using the supernatant.
PO, PPO, and PAL assay PO activity was measured using the method described earlier [49]. The reaction mixture was composed of 0.5 mL of enzyme extract, 1.5 mL of 0.05 M pyrogallol, and 0.5 mL of 1% H 2 O 2 . The reaction mixture was incubated at 28 ± 2 o C for 3 min, and the change in absorbance was recorded at 420 nm every 30 s. The boiled enzyme was added to the test mixture as a blank. PO activity was measured as a change in OD per minute per gram fresh weight. PPO activity was assessed using the method described by Park et al. [50].
Around 200 µL of enzyme extract and 1.5 mL of 0.1 M sodium phosphate buffer (pH 6.5) were used in the test. The reaction was started by adding 200 µL of 0.01 M catechol and the absorbance at 495 nm was measured. PPO activity was measured as a change in OD per minute per gram fresh weight. The PAL assay was carried out exactly as stated by Lee et al. [51]. The assay mixture was made up of 100 µL of enzyme extract, 500 µL of 50 mM tris-HCl (pH 8.8), and 600 µL of 1 mM L-phenylalanine, and it was incubated for 1 h. To stop the process, 2 N HCl was added. The mixture was then vortexed and centrifuged after 1.5 mL of toluene was added (1,000 rpm, 5 min). After separating the toluene phase, the trans cinnamic acid level was measured at 290 nm with a spectrophotometer (Systronics®, Specto-photometer166). A standard was prepared by using cinnamic acid in toluene. PAL activity is expressed as trans cinnamic acid per hour per gram fresh weight.

Statistical analysis
Analysis of variance was used to analyse all the data and the mean values were compared using Tukey's honest significant difference test. The figures were created with the statistical software R and Origin pro. The PRIMER 7 programme was used to create the dendrograms.

Screening of mutants
After gamma irradiation, 250 colonies were randomly picked for each antagonistic bacterial strain based on colony morphology (Figure 1). As a result, in a dual plate assay, all of the selected colonies were tested for antifungal activity against M. phaseolina and F. oxysporum sp. udum. The results revealed that mutant strains had stronger inhibitory action than their wild-type counterparts. The rate of inhibitory activity was found to be higher at 2,500 and 3,000 Gray radiation doses. M. phaseolina was inhibited by B. siamensis BRBac 21-M10 to a maximum of 69%. F. oxysporum sp. udum was inhibited by B. siamensis BRBac 21-M15 to a maximum of 56%. Also in S. cavourensis BRAcB10 M2 mutant showed the increased inhibition activities against the tested pathogens ( Table 2; Figures S1 and S2).

Analysis of rep-PCR, RAPD fingerprint patterns, and lipopeptide genes
Six primers were utilised to amplify the selected mutants and their wild types for fingerprinting analysis. The banding pattern of each primer differed from one another. In the mutants of B. subtilis BRBac4 and B. siamensis BRBac21, the BOX1AR showed four different types of bands ranging from 800 to ∼2,500 bp ( Figure 2). The identical BOX1AR primer, however, produced nine distinct bands in the S.
In this study, all of the primers examined exhibited a considerable amount of polymorphism in all of the strains tested. Furthermore, the presence and disappearance of bands was revealed to be a clear difference between the RAPD and rep-PCR fingerprint profiles of wild and mutant colonies. For example, the BOXA1R primer showed only four bands in BRBac4 except the M8 mutant, it showed the additional 5 bands in ∼1,000 to ∼1,500 bp. Similarly, in the BRBac21, two mutants showed additional bands in ∼150, ∼250, and ∼450 bp. Likewise in the BRAcB10, two mutants showed a higher intensity of few extra bands compared to its wild types. The ERIC 1R primer, bands were observed with the molecular weight of 200, ∼550, 600, 1,000, 1,500, and one additional band at ∼2,500 bp which was absent in mutants M10 and M11 of BRBac4. In BRBac21, ∼2,500 bp amplicon was absent in mutants while it was noticed in wild type. Also in BRAcB10, ERIC 1R primer showed the 11 bands with some additional bands at ∼650 and ∼1,200 bp were also noticed in mutants which were not observed in wildtype strains. The band with a molecular weight of 700 bp, on the other hand, was missing in BRAcB10 mutants. Amplification of lipopeptide genes such as iturin A (∼650 bp) and surfaction (∼440 bp) from BRBac4 and BRBac21 isolates was confirmed.

Cluster analysis of the rep-PCR and RAPD fingerprints
To separate the wild-type strains and their mutants, cluster analyses were done using data from rep-PCR and RAPD primers. The resulting dendrogram revealed a 91% similarity level between the mutants of B. subtilis BRBac4 and their wild type (Figure 3). Cluster I contained 50% of the isolates, together with the wild type; the remaining mutants belonged to Cluster II. Dendrogram results for B. siamensis BRBac21 and its mutants demonstrated 92% similarity. Cluster I included just two mutants of BRBac21, M11 and M12. Cluster II contained the remaining mutants and wild types. Similarly, the dendrogram of S. cavourensis BRAcB10 and its mutants showed 73% similarity, with two clusters. Cluster I accounted for 41.66% of the mutants among the two clusters. Cluster II contained the remaining 58.3% mutants and wild-type strains (Figure 3).

In vitro screening for plant growthpromoting traits
Among the mutants studied, B. subtilis BRBac4-M8 had the highest phosphate solubilisation efficiency (182.45%), followed by BRBac4-M6 (179.12%) and BRBac21-M10 had the lowest (153.25%) ( Table 1). All Bacillus and Streptomyces mutants had an orange clear zone around the colonies, indicating that they were producing siderophores. When compared to wild types, mutants produced significantly less IAA. The highest level of IAA was found in B. subtilis BRBac 4 (16.4 µg mL −1 ), while the lowest amount was found in S. cavourensis BRAcB10-M4 (3.1 µg mL −1 ) ( Table 1). In 0.3% agar containing LB medium, all wild and mutant Bacillus showed greater rates of swimming motility; however, in swarming motility B. subtilis BRBac4-M6 only exhibited the minimal range of swarming motility ( Figure S5).

Antagonistic effects on wilt and root rot disease
The disease symptoms were discovered after the plant had germinated.  Figure 4). Red gram treated with F. oxysporum f. sp. udum exhibited wilt symptoms. Compared to root rot, the maximum wilt incidence (90.2%) was noticed in pathogen inoculated control, followed by T1 (53%) and T3 (45%).

Discussion
Strain improvement is essential during the natural product commercialisation process, as the low titer of wild- type strains can never match the criteria of industrial development and sales [52]. The "random mutation and screening" technique has proven itself numerous times over the last century, making it the best option for strain enhancement today and in the foreseeable future. Mutation is a commonly utilised method in beneficial microorganisms for strain development in order to increase secondary metabolite production and antagonistic activity against diverse pathogens [53]. Ethyl methanesulphonate, hydroxylamine, methyl methanesulphonate, and N-methyl-N-nitro-N-nitroso guanidine are the most commonly used chemical mutagens. Physical approaches, like UV and gamma irradiation, can also be used to induce mutations [54]. The most popular way for obtaining high yielding mutants is to treat a population with a mutagenic agent until a specific "desired" kill is attained, plate out the survivors, and then test each resulting colony or a randomly selected group of colonies in flasks for product creation [30].
Random mutation was conducted in this study using gamma irradiation at different doses ranging from 0.5 to 3 kGy for both Bacillus and Streptomyces. The results showed that greater gamma-irradiated doses, such as 2.5 and 3 kGy, had a higher level of antifungal action. At dosages of 2, 2.5, and 3 kGy, antagonistic mutants of B. subtilis BRBac4-M4, BRBac4-M6, BRBac4-M8, B. siamensis BRBac21-2, BRBac21-10, BRBac21-15, and S. cavourensis BRAcB10-M2 were produced. These findings are consistent with those of Afsharmanesh et al. [33], who discovered that gamma-irradiated Bacillus subtilis UTB1 mutants increased biosurfactant synthesis and control of Aspergillus flavus. Trichoderma virens treated with gamma rays produced two to three times as many secondary metabolites [55]. Ion beam implantation resulted in increased surfactin synthesis in B. subtilis E8 [56]. Gamma irradiation was also used to boost antibiotic paromomycin synthesis in S. rimosus subsp. paromomycinus. In addition, when compared with natural strains, this mutant produced 1.4-2 times the amount of paromomycin [38]. Through the DNA repair systems within cells, gamma ray irradiation may cause certain mutations in cell genes [57,58]. Following 250 Gy of gamma irradiation, C/G base repair substitutions were the most common form of gamma ray induced mutations in Escherichia coli and spontaneous mutational hot spots around positions 620-632 in the lacI gene were reduced [59]. Gamma irradiation may have also boosted the expression of genes responsible for lipopeptides, antifungal metabolites, and plant growth-boosting characteristics in this study. However, it will need to be verified in the future using whole genome sequencing and transcriptome analysis.  Genomic rep-PCR fingerprinting is a technique for identifying and classifying bacteria. This technique is highly reliable, reproducible, and fast [60], and it is utilised to disclose the molecular diversity in the bacterial genome. As a result, this approach was used to determine the genome modification after gamma irradiation in contrast to wild strains. Afsharmanesh et al. [32] used rep-PCR fingerprinting to compare the genomic diversity and polymorphic bands of Bacillus subtilis UTB1 gamma-irradiated mutants with wild-type strains. Abbasi et al. [34], like us, used rep-PCR and RAPD techniques to identify variance in natural and gamma-induced mutant strains of T. harzianum. Several investigations have found that gamma-irradiation can change enzyme activity and secondary metabolites in microorganisms [61,62]. Soufi et al. [35] used rep-PCR (ERIC and BOX) to distinguish the gamma mutation from wild Trichoderma aureoviride strains. T. aureoviride mutants Tv3, Tv2, Tv6, Tv4, and Tv23 showed no genetic resemblance to wild and significantly improved biocontrol abilities in plate experiment. Gomaa and El-Mahdy [40] used gamma mutation to enhance chitinase output in B. thuringiensis NM101-19 and the mutants were screened using the RAPD and inter simple sequence repeat techniques. Rep-PCR (ERIC, BOX) and RAPD primers were examined for B. subtilis BRBac4, B. siamensis BRBac21, and S. cavourensis BRAcB10 and its mutants, resulting in significant differences in the wild type and mutant genomes. In B. subtilis BRBac4-M8, box primer produced bands at 450, 1,000, 1,200, and 1,300 bp, separating this mutant from its wild type (Figure 2). B. siamensis BRBac21-M4 exhibited extra bands of 150, 250, and 400 bp. These bands are most likely associated to the genes that control the expression of mycolytic enzymes, although more research is needed to prove this. Bacillus and Pseudomonas species produce lipopeptides which are antibacterial substances [63,64]. Surfactin, fengycin, and iturin are the three primary lipopeptides secreted by Bacillus spp. [65,66]. Surfactin and iturin genes were found in both B. subtilis BRBac4 and B. siamensis BRBac21 in this investigation. This is consistent with the findings of Gond et al. [42], who discovered the iturin gene as well as additional lipopeptide genes in B. subtilis.
In this study, pot culture data demonstrated that biocontrol agents inoculated treatments had longer root and shoot lengths than controls. Plant development was considerably increased by plant growth promoting rhizobacteria (PGPR) characteristics through both direct and indirect routes. According to Mehmood et al. [67], the use of biocontrol chemicals improved seed germination, root and shoot lengths, and dry biomass. Siderophore producing B. subtilis reduced the frequency of F. oxysporum wilt in cotton and also demonstrated numerous PGPR features such as IAA, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, and phosphate solubilisation [68]. Several studies found that Bacillus spp. and Streptomyces isolated from the rhizosphere demonstrated phosphate solubilisation and antagonism against Fusarium spp. and other phytopathogens [69][70][71]. In chickpea and lentil, a mutant of T. virens outperformed natural strains in terms of antagonistic capacity against Sclerotium rolfsii. Mukherjee et al. and Yandigeri et al. [55,72] revealed that numerous strains of Streptomyces produced more IAA, which boosted wheat crop growth and yield. Bacillus amyloliquefaciens isolated from potato rhizosphere soil showed increased IAA and phosphate solubilisation efficiency, as well as antagonistic activity against Rhizoctonia solani and F. solani. Various Bacillus spp. exhibited ACC deaminase activity, which has a significant impact on plant development and biochemical properties [73,74]. Many actinobacterial taxa with ACC deaminase activity have been identified, including Streptomyces, Amycolatopsis, Mycobacterium, Rhodococcus, and Nocardia [75]. Hydrolytic enzymes (such as cellulase, protease, chitinases, amylase, lipase, and pectinase) have enhanced the PGPR properties associated with the control of plant fungal infections via cell wall disintegration [76]. Our strains also tested positive for a variety of hydrolytic enzymes. Several investigations have found that Bacillus spp. [8,77,78] and Streptomyces [79,80] produce mycolytic enzymes that efficiently suppress fungal infections.
Several previous studies [81,82] found that VOC might inhibit fungal infections. B. subtilis and Actinomyces produce VOCs such as alcohols, ketones, aldehydes, acids, hydrocarbons, and S-containing chemicals [62,80]. In the B. subtilis BRBac4-M6, B. siamensis BRBac21-M10, and S. cavourensis BRAcB10-M2 mutants, approximately 26 compounds were discovered. VOC chemicals generated by mutants demonstrated similarity to their wild-type counterparts (unpublished data). B. siamensis BRBac21 wild strain generated 31.4% Furan, 2-methyl, which was also observed in the BRBac21-10 mutant (28%). Similarly, 4.8 and 6% of 1-pentadiene were found in BRBac21 and its mutant BRBac21-10, respectively. When mutants are compared with wild strains, certain novel chemicals are discovered (Table S2). When compared to the control, the activity of plant defence enzymes was relatively elevated in all treatments. Similar to these findings, other studies have found enhanced production of defence enzymes such as PAL, PO, and PPO in bacterial inoculation treatments compared with controls [8,83,84]. Many studies have found that the PGPR aids in plant defence response to pathogen invasion by possibly increasing chitinase activity, siderophore synthesis, and protease activity [85][86][87]. In this study, highly effective mutants due to gamma irradiation, B. siamensis BRBac21-M10 and S. cavourensis BRAcB10-M2, were chosen for investigation based on their enhanced antifungal activity against M. phaseolina and F. oxysporum f. sp. udum.

Conclusion
Mutant Bacillus and Streptomyces strains displayed significantly better antagonistic activity against M. phaseolina and F. oxysporum f. sp. udum compared with wild-type bacteria, owing to increased synthesis of lipopeptides, hydrolytic enzymes, and swarming and swimming motilities. Analyses of DNA fingerprinting revealed significant polymorphisms between wild type and mutant strains. The combined inoculation of B. siamensis BRBac21-M10 + S. cavourensis BRAcB10-M2 resulted in the lowest incidence of root rot and wilt disease. In addition, the same treatment increased the activity of the defence enzymes PO, PPO, and PAL in pot culture conditions, indicating that, in addition to direct antagonism, these bacteria generated systemic resistance in pulses. These findings suggested that gamma-induced mutation can be used effectively to improve the biocontrol characteristics of Bacillus and Streptomyces. In conclusion, after field testing in multiple places, a combination bio-formulation of these two rhizobacteria may be used to address wilt and root rot pathogens in pulses.