Boosting the Biocontrol Efficacy of Bacillus amyloliquefaciens DSBA-11 through Physical and Chemical Mutagens to Control Bacterial Wilt Disease of Tomato Caused by Ralstonia solanacearum

Bacterial wilt disease of tomato (Solanum lycopersicum L.), incited by Ralstonia solanacearum (Smith), is a serious agricultural problem in India. In this investigation, chemical mutagenic agents (NTG and HNO2 treatment) and ultraviolet (UV) irradiation have been used to enhance the antagonistic property of Bacillus amyloliquefaciens DSBA-11 against R. solanacearum UTT-25 towards an effective management of tomato wilt disease. The investigation established the fact that maximum inhibition to R. solanacearum UTT-25 was exerted by the derivative strain MHNO2-20 treated with nitrous acid (HNO2) and then by the derivative strain MNTG-21 treated with NTG. The exertion was significantly higher than that of the parent B. amyloliquefaciens DSBA-11. These two potential derivatives viz. MNTG-21, MHNO2-20 along with MUV-19, and a wild derivative strain of B. amyloliquefaciens i.e.,DSBA-11 were selected for GC/MS analysis. Through this analysis 18 major compounds were detected. Among the compounds thus detected, the compound 3-isobutyl hexahydropyrrolo (1,2), pyrazine-1,4-dione (4.67%) was at maximum proportion in the variant MHNO2-20 at higher retention time (RT) of 43.19 s. Bio-efficacy assessment observed a record of minimum intensity (9.28%) in wilt disease and the highest bio-control (88.75%) in derivative strain MHNO2-20-treated plants after 30 days of inoculation. The derivative strain MHNO2-20, developed by treating B. amyloliquefaciens with nitrous acid (HNO2), was therefore found to have a higher bio-efficacy to control bacterial wilt disease of tomato under glasshouse conditions than a wild-type strain.


Introduction
Tomato (Solanum lycopersicon L.) is one of the most important solanaceous crops which is prone to bacterial wilt disease caused by Ralstonia solanacearum (Smith),a soil borne pathogen, under favorable conditions, Yabuuchi et al. [1]. The pathogen has over 450 hosts distributed among 54 botanical families and has inflicted heavy yield losses upon various crops worldwide [2].The rate of occurrence of this disease is from 2-60% [3], and the percentage of loss in the yield is 10-92% in tomato plants in India [4,5]. Several management strategies, such as crop sanitation and crop rotation, applying agrochemicals; tube (15 W, 2537 A) at 320 nm and the bacterial cell suspension was distributed in 9 cm autoclaved Petri plates (5 mL in each plate), kept at the distance of 30 cm from the UV source and then exposed to UV radiation for a period of 10, 20 and 30 min intermittently. After exposure, 100 µL of exposed sample was taken from each treatment and inoculated on to the LB-medium containing Petri plates and incubated at 37 • C for 72 h. Individual colonies of variant strain were selected for the screening of an inhibition zone against R. solanacearum.

Chemical Treatment
Forty-eight-hour-old culture containing 10 8 cfu/mL of B. amyloliquefaciens DSBA-11 was used to develop chemical-based mutagenesis as described by Radha Krishnan et al. [10]. Chemical mutagens i.e., NTG (N-methyl-N-nitro-N-nitroso guanidine) and nitrous acid (HNO 2 ) at 50, 100, 150, 200, 250 and 500 µg/mL concentrations were added separately into 5.0 mL of liquid culture of B. amyloliquefaciens DSBA-11 and incubated at 37 • C for 30 min on a rotary shaker at 150 rpm in an Orbital shaker incubator (Model-LOM-560, Make-MRC Laboratory Instruments, Jena, Germany). The incubated bacterial mixtures were centrifuged at 5000 rpm for 15 min; we then discarded the supernatant, and washed twice with the phosphate buffer (pH 6.0).The pellet was then suspended into 10.0 mL of sterile distilled water, re-centrifuged thrice on the same conditions, washed carefully and added with 10.0 mL of saline water (0.85% of NaCl). A 100 µL sample of treated bacterial suspension of each treatment was spread on Luria Bertani (LB) agar medium containing Petri plates in triplicate and incubated at 37 • C for 72 h in the Orbital shaker incubator. The death rate of physical as well as chemical mutagenesis was calculated by the formula I% = [1 − (W t /W 0 )] * 100% where, I represents death rate, W t represents the number of colonies in the UV irradiation group and W 0 represents the number of colonies in the blank group [10].

Antagonistic Ability of Developed Derivative Strains of B. amyloliquefaciens against R. solanacearum
To test antagonistic ability of the developed derivative strains of B. amyloliquefaciens DSBA-11 against R. solanacearum under in vitro conditions as described by Singh et al. [9], the dual culture method was used. The selected derivative strain colonies were grown in Luria broth medium for 4 h at 37 • C and maintained the population of bacteria (0.1 OD at 600 nm). A 100 µL of 48 h old liquid culture (6.2 × 10 8 cfu/mL) of R. solanacearum UTT-25 was spread onto the Petri plates containing the casein peptone glucose agar (CPG) medium separately. Three wells of the diameter 0.5 cm in each Petri plate was made by sterilized cork borer. About 30 µL of derivative strains and wild strain DSBA-11of B. amyloliquefaciens were poured in each well separately with three replications. The Petri plates were incubated at 37 • C for 48 h and inhibition zone was recorded. The value of the inhibition zone was converted into an area of inhibition zone using the formula: Area of inhibition zone = πr 2 [9].

GC/MS Analysis
The three potential antagonistic derivative strains i.e., MUV-19, MNTG-21, MHNO 2 -20 of B. amyloliquefaciens along with wild strain B. amyloliquefaciens DSBA-11 were selected for GC/MS analysis. One liter of 48 h old culture of these strains grown in Luria broth medium was fractioned with the ethyl acetate (500 mL × 3) separately. The ethyl acetate layer was dried over the anhydrous sodium sulphate and under reduced pressure at 45 • C it was collected and dried. Metabolite profiles of the ethyl acetate extract were determined by using gas chromatography and mass spectrometry (Focus-DSQ, Thermo Scientifc, New Delhi, India) equipped with a DB-5 capillary column of the size of 30 m × 0.25 mm and thefilm of thickness of 0.25 µm. Conditions observed while using gas chromatography were: 1. helium was used as the carrier gas with the flow rate of 1 mL/min (split mode, 1:20); injection with the volume 1.0 µL (10 mg extract/3 mL acetone); and column temperature Microorganisms 2023, 11, 1790 4 of 15 maintained at 60 • C and then programmed at 3 • C/min to 280 • C for 5 min. The injector ion source and mass spectrometric transfer line temperatures were kept at 250, 230 and 280 • C. The column was coupled directly to a quadruple mass spectrometer (EI mode, at 70 eV) with the mass range 28-500 a.m.u at 1 scan/s. The compounds were individually identified by comparing their mass spectrum with the spectrum of the compound available in the NIST Mass Spectral Library and literature [21].

Motility of Derivative and Wild Strains of B. amyloliquefaciens
Luria Bertoni agar (LA) and nutrient agar (NA) media containing 1.0 and 1.5 % concentration of agar powder, respectively, with three replications were used to study the motility rate in terms of colony growth of derivative strains MUV-19, MNTG-21, MHNO 2 -20 and wild strain DSBA-11 at 24 h intervals up to 96 h. The fresh colonies of these strains were then placed at the center of the Petri plate and incubated at 30 • C for 96 h. The diameter (in centimeters) of the bacterial colony, thus established, was measured at 24 h intervals.

Bacterial Wilt Disease Control of Tomato by Using Derivative and Wild Strains of B. amyloliquefaciens
The three best potential derivative strains viz., MUV-19, MNTG-21, MHNO 2 -20 and the wild strain DSBA-11 of B. amyloliquefaciens were used to study the bio-efficacy against bacterial wilt and plant-growth-promoting activities in tomato cv. Pusa Ruby under controlled conditions provided by the National Phytotron Facility, IARI, New Delhi, India. Twenty-one-day-old seedlings of tomato cv. Pusa Ruby were transplanted in the pots of 15.0 cm diameter which contained an autoclaved soil mixture of peat moss, vermiculite and sand in the ratio 2:1:1 at 25-30 • C, as well as 48 h old colonies of R. solanacearum and derivative strains along with wild strain DSBA-11 of B. amyloliquefaciens containing bacterial population of 0.1 OD at 600 nm measured by spectrophotometer. After five days of transplanting five seedlings into each pot with three replications, 5.0 mL of liquid culture of R. solanacearum UTT-25 was inoculated at the root zone of the plants. Subsequently, the same amount of derivative strains and wild strain DSBA-11were inoculated at the root zone of the plants separately. The plants treated with R. solanacearum only, and sterilized distilled water-inoculated plants were also maintained to serve as a positive and negative control with three replications, respectively. The observations were recorded at sevendays of intervals up to 28 days after inoculation. The percentage (1-100%) for wilt disease intensity was recorded at the initial stage and final stage (the whole plant wilted). Disease rating was also recorded by using a1-5 scale and wilt intensity was measured as described by Schaad et al. [22]. The biological control efficacy (BCE) of antagonistic bacteria was determined as described by Guo et al. [23].The whole plants with roots were uprooted from each treatment with three replicates. The root and shoot of each plant were cut from the crown region for length (cm) measurement, and fresh weight and dry weight (60 • C for 3 days) were taken. The growth promotion efficacy (GPE) of B. amyloliquefaciens based on plant dry weight was calculated as described by Singh et al. [9].

Statistical Analysis
The analysis of the variance for the biocontrol efficiency and yield of tomato was completed by the SAS general linear model (GLM) procedure (SAS Institute, Version 6, Cary, NC, USA). The mean comparison was conducted by the least significant difference (LSD) test (p = 0.05). Standard error and LSD results were recorded.

Motility of Derivative and Wild Strains
Three derivative strains viz., MUV-19, MNTG-21, MHNO2-20 and wild strain DSBA-11 were streaked on nutrient agar and LA-media containing 1.0 and 1.5% agar and allowed to grow for 96 h. The derivative strains showed faster growth than wild strain DSBA-11 on both the solid media. Composition of medium and agar powder concentrations affected the mobility of the bacteria significantly. LA-medium supported better growth of wild strain DSBA-11 and derivative strains than NA medium. However, LA medium @ 1.0% conc. of agar was found to be the best-suited medium for the growth of derivative strains as well as wild strain DSBA-11 after 96 h of incubation. The maximum colony diameter was found in MUV-19 (7.15 cm) followed by MHNO2-20 (5.76 cm) and MNTG-21 (5.35 cm), which were significantly higher than for wild strain DSBA-11 (4.92 cm) after 96 h of incubation ( Table 4).The box plot represents the motility of B. amyloliquefaciens DSBA-11 and its derivative strains on different media at different concentrations of agar powder ( Figures 3A,B and 4).

Motility of Derivative and Wild Strains
Three derivative strains viz., MUV-19, MNTG-21, MHNO 2 -20 and wild strain DSBA-11 were streaked on nutrient agar and LA-media containing 1.0 and 1.5% agar and allowed to grow for 96 h. The derivative strains showed faster growth than wild strain DSBA-11 on both the solid media. Composition of medium and agar powder concentrations affected the mobility of the bacteria significantly. LA-medium supported better growth of wild strain DSBA-11 and derivative strains than NA medium. However, LA medium @ 1.0% conc. of agar was found to be the best-suited medium for the growth of derivative strains as well as wild strain DSBA-11 after 96 h of incubation. The maximum colony diameter was found in MUV-19 (7.15 cm) followed by MHNO 2 -20 (5.76 cm) and MNTG-21 (5.35 cm), which were significantly higher than for wild strain DSBA-11 (4.92 cm) after 96 h of incubation (Table 4).The box plot represents the motility of B. amyloliquefaciens DSBA-11 and its derivative strains on different media at different concentrations of agar powder ( Figures 3A,B and 4). Table 4. Motility of B. amyloliquefaciens DSBA-11 and its derivative strains on the different media and agar powder concentrations.

DSBA-11 MUV 19 MNTG-21 MHNO 2 -20
Luria agar (1%)      The mean comparison was conducted by the least significant difference (LSD) test (p = 0.05). Different letters point out significant differences in a column. Data present means of the experiment within 3 replications each.   The mean comparison was conducted by the least significant difference (LSD) test (p = 0.05). Different letters point out significant differences in a column. Data present means of the experiment within 3 replications each.

Discussion
Bacterial wilt caused by R. solanacearum is a serious threat to tomato crops in disease-prone areas across the world due to its soil-borne nature and wide host range [10,24]. Management of bacterial wilt disease with microbes, including antagonistic fungi and bacteria, is an alternative technique that is safe, environmentally benign, less expensive, and more sustainable [7,25,26]. In this investigation, we employed B. amyloliquefaciens DSBA-11, a possible antagonistic bacterium used to manage tomato bacterial wilt disease [7,27] to improve its antagonistic capacity and growth boosting activity. The UV irradiation (15 W, 2537 A at 320 nm for 10-30 min of exposure duration) and chemical mutagens such as NTG and HNO2 at concentrations of 50-500 µg/mL were employed to create derivative strains in mutagenesis bacteria [28]. In Pseudomonas fluorescens [10] and

Discussion
Bacterial wilt caused by R. solanacearum is a serious threat to tomato crops in diseaseprone areas across the world due to its soil-borne nature and wide host range [10,24]. Management of bacterial wilt disease with microbes, including antagonistic fungi and bacteria, is an alternative technique that is safe, environmentally benign, less expensive, and more sustainable [7,25,26]. In this investigation, we employed B. amyloliquefaciens DSBA-11, a possible antagonistic bacterium used to manage tomato bacterial wilt disease [7,27] to improve its antagonistic capacity and growth boosting activity. The UV irradiation (15 W, 2537 A at 320 nm for 10-30 min of exposure duration) and chemical mutagens such as NTG and HNO 2 at concentrations of 50-500 µg/mL were employed to create derivative strains in mutagenesis bacteria [28]. In Pseudomonas fluorescens [10] and Bacillus sp. utilized a similar strategy of UV irradiation for the production of derivative strains, whereas Szafraniec et al. [19] and Karanam et al. [13] in Bacillus sp. used chemical mutagens. In the present study, the lethality rate of B. amyloliquefaciens was found to be the highest at 500 µg/mL of both the chemical mutagens i.e., NTG (90.32%) and nitrous acid (95.70%) as compared to UV irradiation (85.75%) at 30 min of exposure. However, the concentration of chemical mutagens and also the exposure duration in UV irradiation decreased the lethality rate. The best three derivative strains, MUV-19 (UV-treated), MNTG-21, and MHNO2-20, demonstrated the strongest antagonistic activity against R. solanacearum in each category of mutagenic agents. However, the maximum inhibition zone of 4.6 cm 2 was formed by MHNO 2 -20 (obtained from nitrous acid treatment at 500 µg/mL) followed by, 3.56 cm 2 by MNTG-21 (NTG-treated) and 3.42 cm 2 by the strain MUV-19 (30 min exposure time), which was significantly higher than the wild strain DSBA-11 (3.35 cm 2 ). The outcomes showed that the bioefficacy of the B. amyloliquefaciens developed derivative strains against R. solanacearum had significantly increased [28], where they used UV light to improve the antibiosis of P. fluorescens (including phenazine, pyrrolnitrin, and phloroglucinol) and siderophores production against damping-off pathogens (Fusarium solani, F. oxysporum, and Rhizoctonia solani). The improvement of actinomycete bacterial strains by using gamma irradiation for enhancing chitinase production activity by induced derivative strain was also reported by Rugthaworn et al. [29] and was found to exhibit a higher inhibitory effect on F. sporotrichioides, R. solani, and Sclerotium rolfsii. Additionally, we employed the wild strain DSBA-11 and three of the most promising derivative strains, MUV-19, MNTG-21, and MHNO 2 -20, in this study to examine the bioefficacy against the bacterial wilt disease in tomato cv. Pusa Ruby under controlled conditions. The results showed wilt intensity in tomato declined significantly (p < 0.05) in MHNO 2 -20-treated plants (Table 5) with the best biocontrol efficacy (88.75) among other treatments. The derivative strain MHNO 2 -20 also promoted plant growth by showing the best growth efficacy (51.79%) followed by wild strain DSBA-11 (27.88%).
We also observed that among the three different mutagens used for development of derivative strains of B. amyloliquefaciens DSBA-11, the derivative MHNO 2 -20 showed significantly highest bioefficacy to inhibit the growth of R. solanacearum and similar result was reported by Haq et al. [30], in which, they found that nitrous acid was found to be the suitable mutagen for improvement of Bacillus spp. Similarly, Sarikaya [11], Haq [31] and Varalakshmi et al. [32] reported that the UV irradiation-treated Bacillus spp. was found most effective in the production of α-amylase. In this study, we observed that the derivative strains developed at lower concentration (50-100 µg/mL) of NGT and nitrous acid and short duration of exposure to UV irradiation showed lesser bioefficacy than wild DSBA-11 (Tables 1 and 2). The similar decreasing trend was also reported in the production of αamylase in a mutant strain of the B. amyloliquefaciens, UNG-16, when compared to the wild strain [33]. Moreover, as per the records of our study, the chance of isolating an effective derivative with the desired trait is lower, in the sense that it was approximately 0.11, 7.0 and 7.4%. Developed derivative strains using UV irradiation, NGT and nitrous acid treatment showed better bioefficacy than wild strain DSBA-11 to form an inhibition zone against R. solanacearum under in vitro conditions. However, under in vivo conditions, out of the three derivative strains used, only the strain MHNO 2 -20 showed a better performance in biocontrol efficacy and plant growth efficacy than the wild strain DSBA-11 (Table 5).
Bacillus spp. secretes various secondary metabolites (including antibiotics, antifungals and siderophores) which can affect the microbiota in the rhizosphere providing an environment antagonistic to pathogens, or trigger host defense responses [34]. In this investigation, a comparative analysis of secondary metabolites produced by derivative strains and wild strain DSBA-11 was analyzed by using GC/MS. Although we found variation in the production of volatile compounds by derivative strains viz., MUV-19, MNTG-21, MHNO 2 -20 and DSBA-11 as mentioned in Table 3, the derivative strain MHNO 2 -20 produced the highest quantity of 3-isobutyl hexahydropyrrolo (1,2-a) pyrazine-1,4-dione and 3-bezylhexahydropyrrolo (1,2-a) pyrazine-1,4-dione while derivative strains MNTG-21 produced diethylphthalate. However, these compounds could not validate against R. solanacearum, though they open the scope of the metabolomics approach in host pathogen interaction studies. Bacillus spp. have ability to extensively colonize the surface of semisolid media by a flagellum independent mechanism and reported that sliding motility is responsible for surface migration [35]. Moreover, the surface colonization is also dependent on the secretion of surfactin, but microscopic examination of the edges and interior cells of sliding surface colonies did not reveal abundant flagella. Fall et al. [36] suggested that B. subtilis has two distinct modes of surface translocation viz., swarming and sliding, which are presumably advantageous under different environmental conditions. In the present study, we observed that motility was comparatively higher in all derivative strains i.e., MUV-19, MHNO 2 -20 and MNTG-21, than in the wild strain DSBA-11 after 96 h ( Table 4). The results showed that the spreading behavior of B. amyloliquefaciens was enhanced on the semi-solid media after bringing it under UV irradiation and then treating with the chemical mutagens. The essential macro-and micronutrients were needed for the sliding motility and the colony spreading as reported by Fall et al. [36] and it was confirmatory to our work. We found that the diameter of the colony of both wild strain and derivative strains was larger on the LA medium as compared to the NA medium after 96 h of incubation at 30 • C ( Figure 5). Further, we found that the level of hardness of the surface of the medium also affected the spreading nature of the bacteria. A 1.0% conc. of agar powder in NA and LA media was found better for the growth of variant strain and parent strain DSBA-11 than a 1.5% conc. of agar because it might have supported swarm or twitching motility of the bacteria on the soft surface of the media in B. subtilis as reported earlier by Fall et al. [36] and in Pseudomonas aeruginosa as reported by Rashid et al. [37]. Ho et al. [38] used the B. amyloliquefaciens strain PMB05 for bacterial wilt disease management, and observed the increase in the signals of Pop W-induced reactive oxygen species generation and callose deposition and thereby confirmed that the PTI was intensified by PMB05.

Conclusions
The following conclusions have been drawn from the results of the study: In comparison to variant strains created by NTG and UV irradiation, the variant strains MHNO2-20 of B. amyloliquefaciens andDSBA-11 were reported to have greater antagonistic potential against R. pseudosolanacearum. It was also observed that, using nitrous acid at 250-500 µg/mL, it was concluded that a high rate of lethality of bacteria treated with chemical and physical mutagens developed a better chance to obtain a potential variant strain with desired traits. Under in vitro circumstances the nitrous acid-treated variant strains were successful particularly in controlling bacteria wilt disease. Additionally, it was observed that chemical mutagens were more efficient than physical mutagens in obtaining the necessary variant strain of B. amyloliquefaciens with better antagonistic ability.