Mining the Genome of Bacillus velezensis VB7 (CP047587) for MAMP Genes and Non-Ribosomal Peptide Synthetase Gene Clusters Conferring Antiviral and Antifungal Activity

Abstract

Chemical pesticides have an immense role in curbing the infection of plant viruses and soil-borne pathogens of high valued crops. However, the usage of chemical pesticides also contributes to the development of resistance among pathogens. Hence, attempts were made in this study to identify a suitable bacterial antagonist for managing viral and fungal pathogens infecting crop plants. Based on our earlier investigations, we identified Bacillus amyloliquefaciens VB7 as a potential antagonist for managing Sclerotinia sclerotiorum infecting carnation, tobacco streak virus infecting cotton and groundnut bud necrosis infecting tomato. Considering the multifaceted action of B. amyloliquefaciens VB7, attempts were made for whole-genome sequencing to assess the antiviral activity against tomato spotted wilt virus infecting chrysanthemum and antifungal action against Fusarium oxysporum f. sp. cubense (Foc). Genome annotation of the isolate B. amyloliquefaciens VB7 was confirmed as B. velezensis VB7 with accession number CP047587. Genome analysis revealed the presence of 9,231,928 reads with an average read length of 149 bp. Assembled genome had 1 contig, with a total length of 3,021,183 bp and an average G+C content of 46.79%. The protein-coding sequences (CDS) in the genome was 3090, transfer RNA (tRNA) genes were 85 with 29 ribosomal RNA (rRNA) genes and 21 repeat regions. The genome of B. velezensis VB7 had 506 hypothetical proteins and 2584 proteins with functional assignments. VB7 genome had the presence of flagellin protein FlaA with 987 nucleotides and translation elongation factor TU (Ef-Tu) with 1191 nucleotides. The identified ORFs were 3911 with 47.22% GC content. Non ribosomal pepide synthetase cluster (NRPS) gene clusters in the genome of VB7, coded for the anti-microbial peptides surfactin, butirosin A/butirosin B, fengycin, difficidin, bacillibactin, bacilysin, and mersacidin the Ripp lanthipeptide. Antiviral action of VB7 was confirmed by suppression of local lesion formation of TSWV in the local lesion host cowpea (Co-7). Moreover, combined application of B. velezensis VB7 with phyto-antiviral principles M. Jalapa and H. cupanioides increased shoot length, shoot diameter, number of flower buds per plant, flower diameter, and fresh weight of chrysanthemum. Further, screening for antifungal action of VB7 expressed antifungal action against Foc in vitro by producing VOC/NVOC compounds, including hexadecanoic acid, linoelaidic acid, octadecanoic acid, clindamycin, formic acid, succinamide, furanone, 4H-pyran, nonanol and oleic acid, contributing to the total suppression of Foc apart from the presence of NRPS gene clusters. Thus, our study confirmed the scope for exploring B. velezensis VB7 on a commercial scale to manage tomato spotted wilt virus, groundnut bud necrosis virus, tobacco streak virus, S. sclerotiorum, and Foc causing panama wilt of banana.

Keywords: B. velezensis; Bacillus amyloliquefaciens VB7; NRPS gene clusters; antifungal action; antiviral action; whole genome sequencing.

Figure 1

Circular Graphical Assembly of the genome of Bacillus velezensis isolate VB7. Note: The outer to inner rings, the contigs, CDS on the forward strand, CDS on the reverse strand, RNA genes, CDS with homology to known anti-microbial resistance genes, CDS with homology to known virulence factors, GC content, and GC skew. The colors of the CDS on the forward and reverse strand indicate the subsystem that these genes belong to (see Subsystems below).

Figure 2

Subsystem Analysis of the Genome Bacillus velezensis-VB7.

Figure 3

Phylogenetic Analysis of the Genome of Bacillus velezensis-VB7 CP047587.

Figure 4

In vitro screening for the antiviral activity of bio-control agents, botanicals, and defense inducers against TSWV on Cowpea (var. Co 7).Note:T1-Bacillus cereus BSC5-0.5% at 10⁸ cfu/mL; T2-B. pumilis BSC4 -0.5% at 10⁸ cfu/mL; T3-B. licheniformis B12-0.5% at 10⁸ cfu/mL; T4-B. velezensis VB7-0.5% at 10⁸ cfu/mL; T5-B. subtilis BAG3-0.5% at 10⁸ cfu/mL; T6-Pseudomonas fluorescens Pf1-0.5% at 10⁸ cfu/mL; T7-Ochrobactrum spp. BSD 5-0.5% at 10⁸ cfu/mL; T8-Mirabilis jalapa (MJ)-10%; T9-Harpulia cupanioides (HC) 10%; T10-Potassium sulphate (K₂SO₄)-0.1%; T11-Potassium silicate (K₂SiO₃)-0.1%; T12-Potassium thiosulphate (K₂S₂O₃)-0.1%; T13-Chitosan-1%; T14-Control.

Figure 5

Antifungal activity of bacterial antagonists against F. oxysporum f.sp. cubense in vitro.