A Novel Rhizospheric Bacterium:

A novel strain of Bacillus isolated from rhizosphere has shown to be an excellent biocontrol agent against various plant pathogens. In this study, a first report of a Bacillus strain NKMV-3 which effectively controls Alternaria solani, which cause the early blight disease in tomato. Based on the cultural and molecular sequencing of 16S rRNA gene sequence, the identity of the strain was confirmed as Bacillus velezensis NKMV-3. The presence of the lipopeptide which are antibiotic synthesis genes, namely iturin C, surfactin A and fengycin B and D, was confirmed through gene amplification. In addition, lipopeptides were also confirmed through liquid chromatography. The extract showed inhibitory effect against A. solani in vitro and detached tomato leaf assays. Bacillus velezensis strain NKMV-3-based formulations may provide an effective solution in controlling early blight disease in tomato and other crops.


Introduction
Tomato (Solanum lycopersicum Linn.) is one of the most important crops cultivated throughout the world in a wide range of climates among solanaceous crops [1,2]. Tomato is ranked as the second most consumed vegetable after potato due to its rich source of vitamin A, C and K, minerals, amino acids, antioxidants and lycopene [2,3]. There is a high demand because of its wide use and nutritional values, both in fresh and processed tomato markets. Therefore, higher production is required to fulfil the increasing demand and for value addition. The tomato cultivation is mainly hindered by bacterial, fungal and viral diseases [4]. The early blight caused by Alternaria solani, in particular, severely affects the tomato production [4,5]. A. solani is an air-borne pathogen inhabiting the soil. It is distributed worldwide but is highly infective in the tropics and temperate regions [5]. Synthetic fungicides such as mancozeb, pyraclostrobin, azoxystrobin and hexaconazole are utilized for the control of this disease in various crops [6,7]. However, A. solani is gaining resistance to these pesticides and causing serious problems in the yield of tomatoes [8,9].
Over the past few decades, chemical pesticides have been extensively used by farmers for crop protection, but it poses severe adverse environmental and detrimental health effects [10,11]. Unscrupulous usage of fungicides encourage in the development of resistant pest/pathogen strains, destruction of non-target organisms, leads to residual toxicity and other health and ecological hazards [12][13][14]. Thus, alternative eco-friendly sustainable agricultural practices like adopting microbial control of pests and diseases are the need of the hour. Bacterial biocontrol agents have recently gained worldwide attention for the control of various plant pathogens due to their low cost and ease of production, environment friendliness and non-residual effect [15,16]. Many species of the genus Bacillus are potential candidates for development as biocontrol agents against various plant pathogens [17,18]. Among Bacillus spp., B. velezensis is one of the potential biocontrol agent [19,20]. Literature reports numerous strains of B. velezensis strains capable of controlling the major plant pathogens (bacteria and fungi) and nematodes [19][20][21][22][23].
In the present study, bacterial strains were isolated from soil samples collected from the roots of tomato plants and screened in vitro for antagonistic activity against A. solani. B. velezensis was identified by 16 s rRNA sequencing. Through FTIR and RP-HPLC analyses, the antifungal lipopeptides secreted by B. velezensis were characterized, and its efficacy against A. solani was studied by poison food technique and detached leaf assays.

Isolation of Bacteria from Tomato Rhizospheric Soil
The sampling was conducted during the months of September to November 2019 in 20 locations of Tamil Nadu known for growing tomato crop in large acreages. Tomato growing fields were selected for sampling soil for the isolation antagonistic bacteria against early blight pathogen, Alternaria solani. Healthy tomato plants were uprooted carefully without disturbing the roots. Soil adhering to the roots was shaken and removed into poly Ziploc bags and immediately kept on ice until further processing.

Isolation of Putative Antagonistic Bacillus Isolates
Soil samples collected from various tomato fields were removed from the cold storage and thawed to room temperature. A soil suspension was prepared using 10 g of each sample and 90 ml of sterile water. The solution was agitated using a rotary shaker at 180 rev min −1 for 30 min. The suspensions thus prepared using all the soil samples were heated to 60 °C for 1 h and cooled immediately by placing in an ice box giving a heat shock reaction for the formation of endospores. The suspensions were serially diluted up to 10 4 and spread plated on nutrient agar medium, followed by incubation at 30 °C for 2 days in an incubator. Single colonies isolated were maintained as purified cultures on nutrient agar slants [24]. Purified putative antagonistic Bacillus isolates were stored at -80 °C in 60% glycerol stocks.

Screening of Putative Antagonistic Bacillus Isolates Against Various Plant Pathogens
Putative antagonistic Bacillus isolates were subjected to screening through dual culture technique [25] against major pathogens of crops, namely Fusarium oxysporum f. sp. lycopersici, Alternaria solani, Rhizoctonia solani, Macrophomina phaseolina and Pyricularia oryzae. All the phytopathogens were supplied by the Microbial Type Culture Collection culture (MTCC) bank, Chandigarh, India. Briefly, 5-mm mycelial plug was removed from an actively growing plate of each phytopathogen and placed at one edge of the plate. Putative Bacillus isolates were streaked on other side of the mycelial plug near the edges of the plate followed by incubation at 28 °C in an incubator for 7-10 days. Bacillus isolates which showed antagonistic activity through zone of inhibitions were selected. The percentage growth inhibition was calculated as per the below formula adapted from [26].
where Control is the colony diameter (cm) in Control plates and Treatment is the colony diameter (cm) in treatment plates. The experiment was performed in triplicates.

Characterization and Identification of Putative Antagonistic Bacillus Isolates
The putative antagonistic Bacillus isolate (NKMV-3) that was shortlisted through dual plate screening was characterized according to Bergey's Manual of Systematic Bacteriology [27] and using Himedia's HiBacillus™ identification Kit. In order to identify the putative antagonistic Bacillus isolate, a loop full culture of NKMV-3 growing on nutrient agar was used for DNA extraction. Quick-DNA Fungal/Bacterial Miniprep Kit of Zymo Research was used for extracting DNA from the putative antagonistic Bacillus isolate (NKMV-3). 16S rRNA sequence of the extracted DNA was amplified through polymerase chain reaction (PCR) with two bacterial universal primers, namely 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) [28]. A 14 µLPCR reaction mixture consisting of 8 µL Taq Master mix, 2 µL of each forward and reverse primers, 2 µL of DNA template and 2 µL of molecular grade water was used for amplifying the DNA. PCR was performed in an Eppendorf Mastercycler X50s. The PCR conditions were adapted from Zhu et al. (2020) with slight modifications as described: 1 min of initial denaturation at 95 ℃, followed by 35 cycles of denaturation at 95 ℃ for 30 s, 1 min of annealing at 52 ℃ followed by extension at 72 ℃ for 2 min and 30 s. A final extension of 72 ℃ for 10 min completed the PCR reaction. The PCR products were purified and sequenced by external sequencing facility. The sequencing results were compared with known bacterial NCBI Genbank sequences using BLAST, and the identity was confirmed. The identified bacterial sequence was submitted with NCBI, and an accession number was obtained. Mega 11 software was used for constructing a phylogenetic tree using the neighbour-joining method with 100 bootstrap replicates [29].

Effect of Crude Lipopeptides Against Alternaria solani
The effect of the crude lipopeptides obtained from NKMV-3 was tested through a modified poison food technique [30]. The dried crude lipopeptide powder was used for preparing various concentrations starting from 1 to 5% in potato dextrose agar medium on W/V basis. Media containing various concentrations of the crude lipopeptides was autoclaved and dispensed in petri plates. After solidification, a 5-mm disc from an actively growing A. solani plate was extracted using a sterile cork borer and placed at the centre of the plate. The PDA plates containing no crude lipopeptides served as control for the trial. The plates were incubated at 28 °C for 7 days in an incubator. After 7 days, the plates were observed for the growth of A. solani. The inhibition rate of mycelial growth was calculated as follows.
The experiment was conducted in triplicates.

Molecular Detection of Surfactin, Fengycin and Iturin genes
A loop full of culture from an actively growing slant of NKMV-3 was used for DNA extraction. DNA was extracted as mentioned in the previous section of this article. Each PCR reaction was performed in an Eppendorf Mastercycler X50s thermal cycler using a 14-µL reaction mixture containing Taq Master mix (8 µL), forward and reverse primers (2 µL each) and molecular grade water (2 µL). The primers were chosen from already available literature. The details of primers and expected amplicon sizes are provided in Table 1. The PCR conditions were adapted from [31,32] with slight modifications. Briefly, SfrA gene was amplified using a 35-cycle reaction consisting of 4 min of initial denaturation  at 95 °C, followed by denaturation for 1 min at 94 °C. Annealing was performed at 52 °C for 30 s, followed by an extension at 70 °C for 1 min. A final extension was performed at 70 °C for 5 min. fen B, fen D and Itu C genes were amplified using a 40-cycle reaction consisting of 3 min of initial denaturation at 94 °C, followed by denaturation for 1 min at 94 °C. Annealing was performed at 59 °C (fen B) and 50 °C (fen D and Itu C) for 1 min, followed by an extension at 70 °C for 1 min. A final extension was performed at 72 °C for 10 min. The PCR amplicons were analysed through gel electrophoresis on a 1% agarose run at constant voltage of 100 V for 40 min followed visualization using a gel documentation system (make: Vilber Quantum).

Extraction of Extracellular Metabolites Produced by NKMV-3
NKMV-3 was grown in Nutrient broth for 72 h at 37 °C in a rotatory shaker with constant shaking of 130 rotations min −1 . The cells were harvested after 3 days by centrifugation at 6,000 × g for 15 min followed by the reduction of pH of the cell free extract to 2.0 by the addition of 3 N HCl and left for overnight precipitation at 4 °C. The so precipitated crude lipopeptides was separated by centrifugation at 8,000 × g for 30 min at 4 °C. The pellet was dissolved in methanol and extracted thrice and evaporated under vacuum using a rotatory evaporator at 50 °C and 65 rpm [33,34]. The resulting viscous liquid was left for drying at 50 °C for 48 h in a hot air oven. The dried crude lipopeptide extract was scrapped and dissolved in Tris HCl pH 7.5 and stored until further use.

Identification and Quantification of Iturin, Surfactin and Fengycin in Crude Lipopeptides using Liquid Chromatography
All solvents used were of HPLC grade (Merck) and standards of iturin, surfactin and fengycin were purchased from Sigma, USA. Crude lipopeptides were quantified by reversed-phase high performance liquid chromatography using a chromatograph (Waters, USA) equipped with a quaternary pump and diode array detector. Analytical scale Purospher® RP-C18 (250 × 4.6 mm, 5 μm particle size) column was used. One mg/ml of crude lipopeptide was prepared and filtered and filled into HPLC vials for injection. A volume of 2 µl of sample was injected into the column. The mobile phase and chromatographic conditions were adapted from [35]. This method provided a single protocol for the detection and quantification of lipopeptides in a combined method rather than the conventional method of separate chromatographic runs for the identification and quantification of individual lipopeptides. The mobile phase consisted of Milli-Q Water (solvent A) and 0.1% HPLC grade trifluoroacetic acid dissolved in acetonitrile (solvent B). The elution of lipopeptide homologues was monitored at 210 nm. The conditions of the chromatography are provided in Table 2.

Analysis of Crude Lipopeptides Through Fourier Transformation Infrared (FTIR) Spectroscopy
The crude methanolic lipopeptide extract was subjected to a FTIR analysis (make:Perkin Elmer) to elucidate the structural groups of the crude lipopeptides. One hundred mg of KBr and a mg of crude lipopeptide extract of NKMV-3 were ground using a pestle and pressed with load for 30 s to obtain translucent pellets. These pellets were subjected to FTIR between a frequency range from 4000 to 400 cm −1 [36].

Detached Leaf Bioassays of Crude Lipopeptides Against A. solani in Tomato
Detached leaf bioassays were conducted with slight modifications as described by [37]. Briefly, tomato (variety: PKM -1) leaves from 45-day-old potted plants were obtained. The leaves were surface sterilized using 1% sodium hypochlorite solution, followed by two washes with sterile water. The leaves were left for air drying inside the laminar air flow chamber. Various concentrations of crude lipopeptides were prepared from 1 to 5% on W/V basis in sterile water and methanol in the ratio of 9:1. Using a handheld atomizer, the leaves were sprayed on both sides with the test solution. Untreated controls were maintained which were sprayed with only sterile water and methanol in the ratios as mentioned above. Control leaves with sterile water spray alone were also maintained. All the leaves were left to air dry inside the laminar air flow chamber. Upon drying, a 5-mm disc from an actively growing 7-day-old A. solani plate was cut and place in the centre of each leaf, except for the untreated control leaves. All the leaves were placed on wet cotton inside petri dishes followed by incubation for 7 days at 28 °C in an incubator. After 7 days, the leaves were examined for lesions, and the extent of lesion formation was measured in centimetres.
The inhibition rate was calculated as follows: The experiment was conducted in triplicates and statistically analysed.

Statistical Analysis
All data was analysed statistically using WASP-Web Agri Stat Package 2.0 and Microsoft Excel (2016) to assess statistically significant differences among the various treatments.

Isolation of Antagonistic Bacteria Against A. solani Causing Early Blight in Tomato
In this study, a total of 146 morphologically distinct isolates of bacteria were isolated from 20 different locations covering three major districts in Tamil Nadu (Salem, Krishnagiri and Dharmapuri) known for the production of Tomato (Table 3). Of the 146 strains screened by dual culture technique (data not shown), NKMV-3 showed maximum inhibition against A. solani. Based on this result, we further explored the biocontrol potential of NKMV-3.

Morphological and Biochemical Characterization of NKMV-3
The cells of the NKMV-3 strain were Gram-positive, motile, short rod-shaped, aerobic bacterium with cell sizes ranging from 1 to 3 µm. The colony of the strain on nutrient agar and Luria Bertani agar was irregular in form with flat elevation and undulate margins. The colour of the colony was dull white with matte texture. NKMV-3 was positive for gelatin liquefaction, catalase and citrate activity. The isolate was able to utilize different sources

Molecular Characterization of 16S rRNA Gene of NKMV-3 Strain
The 16S rRNA gene of strain NKMV-3 was amplified using bacterial universal primers 27F and 1492R. The PCR amplification yielded 1185 nucleotides and sequenced (GenBank accession number: MZ243468). The nucleotide sequence of NKMV-3 16S rRNA gene showed high similarity (99.58%) to the sequence of B. velezensis (Fig. 1). 16S rRNA gene sequence from similar Bacillus species was used for the construction of the phylogenetic tree. NKMV-3 clustered with B. velezensis strains CBMB205 and FZB42.

Antagonistic Effects of B. velezensis Strain NKMV-3 Against Major Phytopathogens
A dual culture technique was performed to evaluate the efficacy of NKMV-3 strain against major agricultural phytopathogens (Fig. 2). After 7 days of incubation, NKMV-3 strain was most effective in inhibiting the mycelial growth of A. solani with an inhibitory percentage of 58.0 ± 0.25%. NKMV-3 was ineffective against Macrophomina phaseolina (Table 5).

In Vitro Effects of Crude Lipopeptides of B. velezensis NKMV-3 Against A. solani
The inhibitory effect of NKMV-3 crude lipopeptide extract was assessed using poison food technique (Fig. 3). It was observed that a 5% crude lipopeptides of NKMV-3 strain significantly (70.8 ± 1.0) inhibited the mycelial growth compared to other tested concentrations ( Table 6).

Amplification of Iturin, Fengycin and Surfactin Genes from B. velezensis NKMV-3
The crude lipopeptides showed inhibitory effect against A. solani, the presence of lipopeptide genes in NKMV-3, namely iturin (ituC), surfactin (sfrA) and fengycin (fenB and fenD), were checked by PCR amplification using primers reported in the literature and as described in the earlier section. Upon PCR amplification, 670 bp corresponding to sfrA, fenB and fenD and 594 bp corresponding to ituC were visualized. Thus, all these four lipopeptide genes were present in B. velezensis NKMV-3 (Fig. 4).

Identification of Lipopeptides from B. velezensis NKMV-3 Though Reversed-Phase HPLC
The lipopeptides, namely iturin, fengycin and surfactin, in crude methanolic extract were separated and identified using a RP-HPLC by comparing its retention time to the specific standards (Sigma, USA). Compounds similar to iturin, fengycin and surfactins were isolated with retention times ranging from 4 to 7 min, 12 to 15 min and 27 to 30 min, respectively (Fig. 5).

Structural Analysis of Crude Lipopeptides from B. velezensis NKMV-3 Through FTIR
The FTIR analysis for crude lipopeptides is exhibited in Fig. 6. A broad peak at 3308 cm −1 specifies the existence of -OH or -NH groups.

Effects of Crude Lipopeptides Against A. solani in Detached Tomato Leaves
The inhibitory effect of various concentrations of NKMV-3 crude lipopeptide against A. solani was assessed using detached tomato leaf bioassay (Fig. 7). There was an evident reduction of lesion diameter with increase in concentration of the crude lipopeptides. Five Values are averages of three replicates. Bars represent means from replicates, and error bars represent standard errors. Different letters indicate significant differences between different treatments according to Tukey's test (α = 0.05).

Discussion
Early blight of tomato has become an economically important disease both under field conditions and also under storage of tomato fruits [38,39], thus garnering interest from both field pathologists and also from post-harvest disease control specialists. Several synthetic fungicides have been used by farmers for the control of early blight of tomato [40]. But, due to their continuous usage and also non-adherence to prescribed dosages, resistance build-up of resistance has been observed [41] among the pathogens. Hence, a safe and sustainable method of disease control is the need of the hour. In the past, both bacterial and fungal biocontrol agents effective against various plant pathogens have been successfully developed and commercialized [42]. Bacterial biocontrol agents had several advantages such as easy to mass multiply, better efficacy, longer shelf life, increased safety to humans and environment in comparison to fungal biocontrol agents. Hence, this study was begun with the objective of isolating native bacteria from tomato growing regions showing antagonistic activity against early blight disease of tomato. Soil samples from 20 different tomato growing regions of the state of Tamil Nadu, India, was used for the isolation of 146 bacterial antagonistic bacteria in this study. Among them, NKMV-3 was found inhibit A. solani with highest percent of inhibition. Many species of the genus Bacillus are known to exhibit antifungal activity against various plant pathogens especially against A. solani [1,43].
Strain NKMV-3 was identified as B. velezensis, based on morphological, biochemical and 16S rRNA gene sequencing. B. velezensis is a farmer friendly bacterium reported to promote plant growth [44], control plant diseases [45,46] and detoxify pollutants [47]. Several B. velezensis isolates were found to effectively control various phytopathogens such as Botrytis cinerea, Alternaria solani, Fusarium oxysporum and Colletotrichum gloeosporioides among others [48]. B. velezensis has already been reported to control R. solanacearum, F. oxysporum and Verticillium dahlia infecting tomato plant [21,49]. This research articles is the first report of a B. velezensis strain isolated from major tomato growing regions of Tamil Nadu, India, which is effective against A. solani.
Dual-culture results showed B. velezensis NKMV-3 not only inhibit the growth of A. solani, but also inhibited other two tomato pathogens, F. oxysporum f. sp. lycopersici and R. solani. These results suggest that B. velezensis NKMV-3 could control not only early blight but also vascular wilt and foot rot diseases in tomato. These were tested at the To further understand the biocontrol mechanism of B. velezensis NKMV-3, A. solani was used as an indicator. We confirmed the presence of essential genes for lipopeptides secretion namely, iturins, fengycin and surfactins in B. velezensis NKMV-3 by PCR amplification. The presence of all the three secreted lipopeptides was identified through RP-HPLC and FTIR analyses. To date, lipopeptides secreted by B. velezensis have been documented to inhibit the growth of phytopathogens, such as R. solani, F. oxysporum, A. flavus and Ralstonia solanacearum [19]. In our findings, 5% crude lipopeptide extract effectively inhibited A. solani as evidenced by both poison plate technique and detached leaf assay. Chen et al. (2018) showed the lipopeptides secreted by B. velezensis LM2303 inhibited F. graminearum by damaging the cell membrane permeability [50]. The same mechanism could also work in A. solani; this will be tested in future.

Conclusion
This study clearly revealed the biocontrol potential of B. velezensis strain NKMV-3 isolated from the tomato growing regions of Tamil Nadu. In vitro investigations through lipopeptide biosynthesis gene detection, RP-HPLC, FTIR analysis and antifungal activities clearly demonstrated its ability to be used as a biocontrol agent for controlling fungal pathogens. The commercialization of the product could be possible with formulations and field trials in future.