Molecular Characterization Reveals Biodiversity and Biopotential of Rhizobacterial Isolates of Bacillus Spp

Bacillus species appearas the most attractive plant growth-promoting rhizobacteria (PGPR) and alternative to synthetic chemical pesticides. The present study examined the antagonistic potential of spore forming-Bacilli isolated from organic farm soil samples of Allahabad, India. Eighty-seven Bacillus strains were isolated and characterized based on their morphological, plant growth promoting traits and molecular characteristics. The diversity analysis used 16S-rDNA, BOX-element, and enterobacterial repetitive intergenic consensus. Two strains, PR30 and PR32, later identified as Bacillus sp., exhibited potent in vitro antagonistic activity against Ralstonia solanaceorum. These isolates produced copious amounts of multiple PGP traits, such as indole-3-acetic acid (40.0 and 54.5 μg/mL), phosphate solubilization index (PSI) (4.4 and 5.3), ammonia, siderophore (3 and 4 cm), and 1-aminocyclopropane-1-carboxylate deaminase (8.1and 9.2 μM/mg//h) and hydrogen cyanide. These isolates were subjected to the antibiotic sensitivity test. The two potent isolates based on the higher antagonistic and the best plant growth-promoting ability were selected for plant growth-promoting response studies in tomatoe, broccoli, and chickpea. In the pot study, Bacillus subtilis (PR30 and PR31) showed significant improvement in seed germination (27–34%), root length (20–50%), shoot length (20–40%), vigor index (50–75%), carotenoid content (0.543–1.733), and lycopene content (2.333–2.646 mg/100 g) in tomato, broccoli, and chickpea. The present study demonstrated the production of multiple plant growth-promoting traits by the isolates and their potential as effective bioinoculants for plant growth promotion and biocontrol of phytopathogens.

Bacterial wilt caused by Ralsatonia solanacearum is a quarantine phytopathogen responsible for devastating agricultural losses worldwide [22].It is a soil-borne phytopathogen that infects various commercial crops and can survive for long periods in the soil.During favorable conditions, the dormant bacterium is activated, enters through primary and secondary roots, and colonizes the host plants' xylem vessels, leading to a lethal wilt disease.This disease causes enormous losses from the time of sowing till maturity.The disease is characterized by the appearance of yellow-colored leaves and light brown-colored lesions on the shoot androot, leading toreduced crop yield [22].
The present study evaluatedthe diversity, plant growthpromoting ability, and antagonistic activity of Bacillus sp.against R. solanacearum in tomato, broccoli, and chickpea.

Screening of Antagonistic Bacillus Sp.
Seven selected Bacillus isolates were used for in vitro antagonistic activity using disc diffusion against R. solanacearum [26].The growth inhibition of the pathogen was measured as the percent growth inhibition (PGI) using the following formulawhere C = measure of control group growth and T = measure of treatment group.
The test was repeated 3 times with 5 independent replications.

Molecular Identification of Bacillus Isolates
The standard microbiological protocols were followed for morphological and biochemical characterization of isolates.DNA extraction, partial 16S rRNA gene amplification, PCR product purification, and subsequent sequencing analysis of Bacillus isolates were performed as previously described [27].The 16S rRNAgenewas amplified using universal primers, PF [5′-TGG CTC AGA TTG AAC GCT GGCGG-3′] and PR [5′-TGG CTC AGA TTG AAC GCT GGCGG-3′], and the PCR products were sequenced on ABI3100 Genetic Analyzer.The amplified sequences were run in the BLASTnprogram and compared with the NCBI database.
Using the multiple sequence alignment tool Clustal W, consensus sequences of the 16S rRNA gene from Bacillus isolates and reference sequences obtained from Genbank were aligned, using MEGA version 5 to contruct a phylogenetic analysis [28].The unweighted pair group technique with the arithmetic mean (UPGMA) approach was used to conduct the analysis.The greatest composite likelihood approach was used to calculate the evolutionary distances, and the evolutionary history was constructed using the neighbor-joining method [29] with 2000 bootstrap replications, and the internal branches' robustness was evaluated.
Molecular identification of Bacillus isolates was performed using rep-PCR with the help of BOXA1R ERIC-1R and ERIC-2F primers [28].The PCR reaction mixture (25 μL) containing 5 × Gitschier buffer, 0.25 μM of primer, 50 ng DNA template, 1 U of Taq DNA polymerase (Bangalore Genie, India), and 2 mM MgCl 2 was amplified on thermal Cycler (Biorad, CA, USA) and the amplified fragments were separated by agarose gel electrophoresis with the help of aDNA ladder of 100 bp to 3 kb.

Indole Acetic Acid Production
Indole acetic acid (IAA) production by the isolates was screened in Luria Bertani (LB) medium containing 1 g/L tryptophan [30].

Phosphate Solubilizing Activity
The inorganic phosphate solubilizing (PS) activities of bacterial isolates were perceived using the National Botanical Research Institute's phosphate growth medium (NBRIP) agar medium at 37 ± 2 °C for 7 days.The abilityof the isolates to solubilize inorganic phosphate was calculated as a solubilization index (SI) using the following formula [31].

Production of Ammonia
Isolates were screened for ammonia (NH 3 ) production in peptone water (10 mL) inoculated and incubated for 48-72 h.Following the incubation, Nestler's reagent

SI =
Colony diameter (mm) + Zone diameter (mm) Colony diameter (mm) (0.5 mL) was added, and the tubes were observed to color change from brown to yellow [32].

Siderophore Production
Screening of isolates for siderophore (SD) production was performed on Chrome Azurol agar (CAS) medium [33] at 30 °C.The isolates were grown on CAS agar medium at 30 °C for 48 h and observed for color change of medium from blue to orange/golden.

Susceptibility to Antibiotics
Bacillus isolates were tested according to the method of Bauer [35] for its resistance to various antibiotics (Table 4).All assays were performed three times with five replications.

In Vitro Study
The seeds of different vegetable crops such as tomato (variety-Lycopersicon; NTL-186), broccoli (variety-PalamSamridhi), and chickpea (variety-Pusa 256) were surface-sterilized with ethanol (70%) for 2 min and washed three times with sterile distilled water.The following treatments were applied.T0-control (no bacterial inoculation), T1 = B. subtilis PR30, T2 = B. subtilis PR31.The surface-sterilized seeds were transferred to bacterial suspension (10 4 cfu/mL), kept for 60 min, placed on germination paper, and maintained for 15 days at 25 °C [24].Control (T0) seeds without bacterial inoculation were used for comparison.Treated and control seeds were also checked for the root and shoot elongation pattern under in vitro conditions.All the treatments were performed in triplicates, and the average of triplicates was considered.

Percent Germination
The percent seed germination was calculated as follows [36].

Germination Index
The germination index (GI) was calculated according to the Association of Official Seed Analysts AOSA [37] using the following formula-

Vigor Index
Vigor index was calculated according to Abdul and Anderson [38] and with the help of the following formula.

Plant Seeds and Treatment
The bioefficacy of Bacillus isolates (PR30 and PR31) was evaluated under a greenhouse pot experiment at Sam Higginbottom University of Agriculture, Technology and Sciences (SHUATS), Allahabad, India.Surface sterilized broccoli, tomato, and chickpea seeds were treated with endospore suspension of Bacillus isolates (~ 10 8 cells mL −1 ).Treated seeds were sown in plastic pots containing autoclaved field soil and watered daily.Control seeds were not treated with bacterial culture.Five seed pot −1 and 10 pot treatment −1 were maintained.The treatments for the bioefficacy experiment were scheduled as (1) B. subtilis PR30, (2) B. subtilis PR31, (3) and Control.Plant growth parameters were measured 30, 60, and 90 days after sowing (DAS).

Measurement of Plant Growth Parameters
Ten seedlings were harvested, root length, shoot length, fresh weight, and dry weight (mg/plant) were measured.The plant height (cm) was measured from the ground level to the growing tip of the main shoot, and the average height was calculated in cm.One plant was randomly selected from each pot, and the number of branches of Vigor index = Percent germination × Seedling length each plant was measured and averaged.Flowers from three plants were counted and averaged.All the fruits from three selected plants from each replication of all the treatments were counted.All the fresh fruits from three selected plants from each replication of all the treatments were weighed after picking.Each replication's average fresh fruit weight per plant (g) was recorded and subjected to statistical analysis.The diameter of the curd of three plants was measured at the widest circumference in cm, and the average diameter per curd from each pot was calculated.
The Head or bud of three selected plants was weighted on electrical balance in g, and the average was found to give the bud weight per plant.The fresh weights of the three selected plants were recorded in each pot, and the average fresh weight was calculated.This calculated value was assumed as the average weight of the rest of the remaining plant per pot.The plants used to measure dry weight were also subjected to dry weight analysis.The plants were dried for 5-6 h at a temperature of 50-60 °C.The dry weights of all randomly selected plants in each pot were added, and the average was calculated.

Biochemical Parameters
Chlorophyll, Carotenoid, and Lycopene Content Chlorophyll a, b, and carotenoid content were determined according to Arnon's method [39].The absorbance of the resulting solution was read at 663, 645, and 480 nm for chlorophyll a, b, and carotenoids.The extraction and estimation of lycopene content was performed according to the method of Butnariuand Giuchici [40].The absorbance of supernatant containing lycopene was read in a spectrophotometer at 472 nm.The total lycopene content was measured as lycopene mg per 100 g of fruit tissues.

Statistical Analysis
All the experiments were performed in triplicates, and the average valueswere calculated.The standard errors were calculated for all mean values and subjected to ANOVA followed by DMRT.The BOX and ERIC-PCR product cluster analysis was based on the binary matrix, presence (1), and absence (0) of the band for each strain.Principle component analysis (PCA) was performed using the XLSTAT software.
Values are the mean of three replicates.According to Duncan's multiple range test, different letters in superscript are significantly different (p ≤ 0.05).

Identification of antagonistic Bacillus isolates
Seven Bacillusisolates were recognized as the active antagonists.Their phylogenetic distribution and 16S rRNA gene sequence individualities are presented in Table 5.The similarity values (≥ 97%) confirmed that all isolates (PR30, PR31, PR33, PR38, PR42, PR45, and PR48) belong to the genus Bacillus subtilis.The un-rooted phylogenetic tree (Fig. 3) presented the genotypic relationship of the isolates, wherever all the bacterial isolates were clustered into two major clades.The 16S-rRNA data showed that PR31and PR35 (B.subtilis) isolate was more diverse than other strains and grouped into distinct clusters.

ERIC and BOX-PCR Analysis
ERIC's complex fingerprint patterns produced 81 polymorphic bands of variable range (250-3000 bp) (Fig. 4a).Principal component analysis (PCA) based on the first and second coordinates showed a maximum Eigen value of 4.642 and minimum value of 0.095 with a percent variation of 66.31 and 14.30, respectively (Fig. 4b).Observation of the PCA analysis revealed that four isolates (PR30, PR31, PR35, and PR55) formed a major cluster (cluster I).Three isolates were classifiedin cluster II (PR38, PR42, and PR45).
The BOX-PCR banding pattern of all seven strains displayed 102 fragments in the 250-4000 bp (Fig. 4c).The results of the PCA analysis are based on the first and second coordinates and showed a maximum Eigen value of 3.053 and minimum value of 0.162 with a percentage  variation of 50.05 and 13.90%, respectively (Fig. 4d).PCA analysis revealed that four isolates (PR30, PR31, PR38, and PR55) formed a major cluster (cluster II), and three isolates (PR35, PR42, and PR45) were classifiedin cluster I.

Evaluation for Bioefficacy
Based on antagonistic activity and PGP traits, two isolates (PR30 and PR31) were selected for plant growth parameters of different vegetable crops under in vitro and greenhouse conditions.The Bacillus isolates (PR30 and PR31) induced a higher percentage (85-96%) of germination of seed compared to control seeds (Table 6).

Discussion
Increasing agricultural productivity with limited cultivable land is the biggest challenge to growers around the globe.It is necessary to improve agricultural productivity to nourish and feed the growing world population.Crop yield and productivity can be enhanced in two ways: by increasing crop productivity through fertilizers or biofertilizers and by preventing crop losses caused by phytopathogens.Using PGPR that possesses the dual potential of plant growthpromotion and biocontrol is expected to play this dual role [24].
The diverse potential of Bacillus spp.makes it a promising plant growth-promoting rhizobacterium and BCA in various crops.Inoculation of crops with Bacillus spp.promotesseed germination, seedling vigor, leaf index, root and shoot growth, and photosynthetic ability.The plant growth promotion due to Bacillus spp.inoculation is due to the production of various PGP substances [2,14].Inhibition of phytopathogens results from the secretion of a wide range of antagonistic substances [17,26].Members of the Bacillus genus produce multiple PGP traits, such as phytohormones, and they help in nutrient mobilization (iron, P, etc.) [7,14], which improves the growth of inoculated crop plants.Bacillus spp. is one of the major biological control agents (BCA) and antagonistic soil bacterium [21,26].Bacillus sp.produces various antagonistic substances, such as hydrogen cyanide, siderophore, and hydrolytic enzymes to inhibit the growth of phytopathogens.
Developing biofertilizers/formulation strategies using spore-forming Bacillusbioagents is an emerging area in crop protection.A total of 87 Bacillus strains were isolated from organic farm soil samples and examined by performing a disc diffusion approach to raise the possibility of using antagonistic bacteria asBCAs against Ralstonia solanacearum.Using this strategy, seven potent B. subtilis strains (PR30, PR31, PR35, PR38, PR42, PR45, and PR55) were specifically selected as an effective biocontrol agent against R. solanacearum.Similar strategies have been used effectively to isolate potential BCAs, such as Bacillus strains exhibiting antimicrobial activity towards phytopathogens [41].
Molecular identification based on 16S rRNA gene sequences of Bacillus isolates indicates phylogenetic clustering between bacteria at inter-and intra-species levels [42].At the same time, the present study perceived that identification based on 16S rRNA gene sequences is limited and incapable of distinguishing along with bacterial strains.Consequently, polyphasic gene-based fingerprinting tools (BOX-PCR and ERIC-PCR] were used to discriminate intraspecies unevenness between the bacterial strains.The results presented that isolates could not be distinguished by partial 16S rRNA gene sequence were different regarding ERIC and BOX-PCR patterns.Besides, these tools help separate the ecologically diverse Bacillus strains into a distinct group, which is otherwise tricky through 16S rRNA gene analysis [23]. Seedling treatment among isolates significantly suppressed the pathogen growth in in vitro conditions and enhanced plant height and biomass compared to control.Inhibition of phytopathogens is attributed to various antagonistic substances,viz.siderophore,hydrogen cyanide, other volatile compounds, and a wide range of antibiotics.Siderophore-producing PGPR prevents iron nutrition and, hence, the growth of phytopathogens [43].HCN is a volatile organic compound (VOC) synthesized by a wide range of PGPR.Many bacterial genera, including Bacillus, can produce HCN [6,20].HCN exerts a potent toxic effect on many phytopathogens, forming stable complexes with Cu 2+ , Fe 2+ , and Mn 2+ and causing disruption in protein functions [25,44] (Reference?).It also inhibits electron transport and disrupts the energy supply to the cell, leading to living organisms' death.Besides their biocontrol ability to produce HCN and antibiotics, Bacillus species produce phytohormones, increase uptake of phosphate and iron, produce ammonia,  Many PGPR produce IAA, a crucial phytohormone associated with root elongation and initiation.Plants provide tryptophan to PGPR to synthesize IAA, an essential phytohormone for plant growth promotion.Bacillus spp.produced copious amounts of IAA, and this IAA has promoted root ramification.
Phosphorus is one of the vital elements for plant growth and development.It is regarded as a limiting nutrient for plant growth as it is usually present in insoluble forms.P solubilizing PGPR can potentially solubilize P and make it available forplant growth promotion [45].
Liu et al. [14] also suggested that the B. amyloliquefaciens strain displays maximum in vitro inhibitory activity towards multiple plant pathogens.Sudha et al. [45] reported the production of volatile compounds in Streptomyces rochei that inhibited the growth of sorghum pathogen.Sayyed and Patel [46] presented siderophore production in Alcaligenesfaecalis and found that this siderophoregenic culture inhibits the growth of a wide range of fungal phytopathogens.They found more antifungal activity in siderophoregenic culture than in a chemical fungicide.The present study also demonstrated that B. subtilis PR31 colonized more frequently than other test strains in tomato and broccoli rhizosphere, while B. subtilis PR30 was assessed more in chickpea.These findings justify the previous biocontrol reports that emphasize the proficient colonization of BCAs in the host rhizosphere,which is expected to enhance plant growth promotion and disease management.
Biocontrol efficacy of Bacillus spp.has been confirmed in the greenhouse and field conditions and at the post-harvest stage for fruit diseases [47].It has been established primarily to resist gram-negative bacteria in vitro and under controlled conditions and to reduce diseases caused by these pathogens.A single strain can act against numerous bacterial pathogens.For example, B. velezensis LS69 has been shown to display antibacterial activities against Erwinia carotovora and Ralstonia solanacearum [43,48].Production of plant growthpromoting traits (PGPT) is the characteristic feature of all PGPR.These PGPT promote plant growth through direct mechanisms as green biostimulants [2,49].PGPR promotes plant growth through an indirect mechanism, such as the production of antibiotics [43] and the production of hydrolytic enzymes.The induction of resistance in plants and production of siderophore [46,50] and phosphate solubilizing ability in different cultures of PGPR isolated from the rhizosphere have been reported.Kapadia et al. [51] reported the production of multiple plant growth-promoting traits in Bacillus sp., Klebsiella variiocola, and Mesorhizobium sp., respectively, and found that this multipotent culture improves growth in wheat and maize.The Bacillus sutilis isolates identified and used in the present study possessed all the plant growth promoting traits and hence are ideal candidates for biological control of R. solanacearum.
The production of ammonia is one of the major traits of PGPR that helps promote plant growth.The production of ACCD in PGPR is one of the best mechanisms involved in plant growth promotion under oxidative stress.PGPR a b c Fig. 5 Plant growth promotion-greenhouse pot assay (5a).Tomato with control, (5b) Broccoli with control, and (5c).Chickpea with control lowers the ACC level in root exudates, decreasing the concentration of ethylene in the plant roots and thus helps in root length for better absorption of nutrients.Through antioxidants and other mechanisms, PGPR induces resistance to protect crop plants, thus helping plant growth [47,48,[52][53][54].
Bacillus spp.suppressing the development of a wide range of phytopathogens while promoting plant growth in various crops can make this culture a multipotent PGPR for sustainable plant disease management and an eco-friendly biocontrol agent [2].
The present study successfully screened the Bacillus strains such as B. subtilis PR30 and B. subtilis PR31 associated with tomato rhizosphere that could stimulate growth in various crops (tomato, chickpea, and broccoli).These strains are identified as potent antagonists to suppress the growth of R. solaniunder in vitro conditions.They can be used in integrated disease management of tomato root rot and damping off.The combined studies, comprising biochemical and molecular technologies, are essential to select indigenous antagonistic Bacillus strains that can be used in combinations of other strains under different environmental conditions (greenhouse and field conditions to obtain resistance against pathogens in various crops).Without the extraordinary effect of genetic resistance in tomato cultivars, these biocontrols may be a potential candidate for handling vascular wilt infection and minimizing losses in enhanced fruit quality and yield.However, additional investigations are required to conclude the efficiency of these strains under diverse cultivar varieties and locations to understand the interaction behavior with the pathogen, host plants, and soil factors.

Fig. 1
Fig. 1 Map showing SHAUTS farm (Courtesy: Google map) Seed germination (%) = Number of germinated seeds Number of total seeds × 100 GI = No. of germinated seed Days of the first count + No. of germinated seed Days of the final count

Fig. 2
Fig. 2 Antagonistic activity of isolated bacteria against R. solanacearum

Table 1
Antagonistic activity of isolated bacteriaagainstRalstonia solanaceorum

Table 2
Morphological and biochemical characterization of antagonistic isolates of Bacillus sp.against R.solanaceorum -It was not determined.Values are the average of three replicates analyzed by Duncan's multiple range test

Table 4
Antibiotic resistance of antagonistic isolates of Bacillus sp

Table 5
Identification of isolated bacteria based on 16S rRNA gene sequencing

Table 6
Inoculation effect of B. subtillis on percent seed germination and growth parameters of vegetable crops under in vitro condition

Table 7
Effect of Bacillus subtilis on growth promotion of vegetable crops under greenhouse conditions

Table 8
Effect of Bacillus subtilis on Biochemical traits of vegetable crops under greenhouse condition