Nematicidal Property of Clindamycin and 5-hydroxy-2-methyl Furfural (HMF) from the Banana Endophyte Bacillus velezensis (YEBBR6) Against Banana Burrowing Nematode Radopholus similis.

Radopholus similis is a burrowing nematode which causes banana toppling disease and is of major economic threat for the banana production. Bacterial endophyte Bacillus velezensis (YEBBR6) produce biomolecules like 5-hydroxy-2-methyl furfural (HMF) and clindamycin in during interaction with Fusarium oxysporum f.sp. cubense. Molecular modelling and docking studies were performed on Radopholus similis protein targets such as calreticulin, cathepsin S-like cysteine proteinase, ß-1,4 -endoglucanase, reticulocalbin, venom allergen-like protein and serine carboxypeptidase to understand the mode of action of HMF and clindamycin against Radopholus similis. Structurally validated protein targets of R. similis were docked with biomolecules through AutoDock Vina module in PyRx 0.8 software to predict the binding energy of ligand and target protein. Among the chosen six targets, docking analysis revealed that clindamycin had the maximum binding affinity for ß-1,4-endoglucanase (- 7.2 kcal/mol), reticulocalbin (- 7.5 kcal/mol) and serine carboxypeptidase (- 6.9 kcal/mol) in comparison with HMF and the nematicide, carbofuran 3G. Besides, clindamycin also had the maximum binding energy for the target sites calreticulin and venom allergen-like protein compared to the small molecule HMF. Novel molecule, clindamycin produced by B. velezensis served as a potential inhibitor of the target sites associated in interrupting the functions of ß-1,4-endoglucanase, reticulocalbin, serine carboxypeptidase, calreticulin, cathepsin S-like cysteine proteinase, and venom allergen-like proteins. Besides, increased binding affinity of clindamycin with the protein target sites facilitated to explore it as a novel nematicidal molecule for the management of banana burrowing nematode R. similis. Thus, present investigation confirmed that, the small molecules clindamycin can be explored for nematicidal activity. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01011-2.

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

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.

Molecular modelling of coat protein of the Groundnut bud necrosis tospovirus and its binding with Squalene as an antiviral agent: In vitro and in silico docking investigations.

Bud blight disease caused by groundnut bud necrosis virus (GBNV) is a serious constraint in the cultivation of agricultural crops such as legumes, tomato, chilies, potato, cotton etc. Owing to the significant damage caused by GBNV, an attempt was made to identify suitable organic antiviral agents through molecular modelling of the nucleocapsid Coat Protein of GBNV; molecular docking and molecular dynamics that disclosed the interaction of the ligands viz., Squalene and Ganoderic acid-A with coat protein of GBNV. Invitro inhibitory effect of Squalene and Ganoderic acid-A was examined in comparison with different concentrations, against GBNV in cowpea plants under glasshouse condition. The different concentrations of Squalene (50, 100, 150, 250 and 500 ppm) tested in vitro resulted in reduction of lesion numbers (1.69 cm2) as well as reduced virus titre in co-inoculation spray. The present study suggests the antiviral activity of Squalene by effectively fitting into binding site of coat protein of GBNV with favourable hydrophilic as well as strong hydrophobic interactions thereby challenging and blocking the binding of viral replication RNA with coat protein and propagation. The present organic antiviral molecules will be helpful in development of suitable eco-friendly formulations to mitigate GBNV infection disease in plants.

Antiviral activity of basidiomycetous fungi against Groundnut bud necrosis virus in tomato.

Tomato is an important vegetable crop which is severely affected by Groundnut bud necrosis virus (GBNV). Until now effective antiviral agents have not been reported for the management of necrosis disease caused by GBNV. Therefore, a study was undertaken to manage the necrosis disease caused by GBNV using culture filtrate of basidiomycetous fungi viz., Coprinopsiscinerea, Ganoderma lucidum and Lentinula edodes. In vitro studies were conducted in the indicator host cowpea and primary host tomato in glasshouse under insect proof condition; co-inoculation spraying of culture filtrate of Ganoderma lucidum at 0.1% concentration reduced the lesion numbers and inhibited the virus population build-up when compared to inoculated control in the indicator host cowpea upto 77.83%. DAC-ELISA test was performed to quantify the virus titre, indicated reduced virus titre in co- inoculation spray of culture filtrate of G. lucidum treated cowpea with OD value 0.17 ± 0.01 at 405 nm and in tomato plants 0.14 ± 0.01 respectively. The viral copy numbers were quantified by qPCR. About 2.0 × 101 viral copy numbers were observed in tomato plants treated with G. lucidum (co-inoculation) which was lesser than untreated inoculated control plants (2.4 × 108). In order to identify the antiviral properties of G. lucidum, GCMS analysis was carried out and we found the triterpenoid compound Squalene. This is the first study to analyse and confirm the antiviral activity of G. lucidum against a plant virus.

Effect of Headspace and Trapped Volatile Organic Compounds (VOCs) of the Chinese Caterpillar Mushroom, Ophiocordyceps sinensis (Ascomycetes), against Soil-Borne Plant Pathogens.

Headspace volatile metabolites produced by Ophiocordyceps sinensis were tested against soil-borne plant pathogens (namely, Fusarium oxysporum f. sp. lycopersici, F. oxysporum f. sp. cubense, Thanatephorus cucumeris, Athelia rolfsii, and Macrophomina phaseolina). Diffusible volatile metabolites produced by O. sinensis inhibited 52% and 48% of the mycelial growth of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. cubense, respectively. In addition to inhibiting mycelial growth, the headspace volatile metabolites also induced several morphological changes in the culture characteristics and mycelia of the tested fungi. Stunted and depressed colony growth was observed for F. oxysporum f. sp. cubense and F. oxysporum f. sp. lycopersici. The headspace volatile compounds produced by O. sinensis were trapped in a glass cartridge (Porapak Q). The trapped compounds were eluted from the column by using hexane and then, by using gas chromatography-mass spectrometry, were identified as tetratetracontane, 1(2H)-naphthalenone, 3, 4-dihydro-3-methyl, 3-hexenoic acid, 1-methyl-3-ethyladamantane, and phenol, 3-ethyl.